Antibodies directed against amyloid-beta peptide and methods using same

ABSTRACT

Monoclonal antibody 9TL and antibodies derived from 9TL directed against amyloid-beta peptide and methods of using same for diagnosing and treatment of Alzheimer&#39;s disease and Aβ peptide associated diseases are described. Methods of using antibodies directed against amyloid-beta peptide having impaired effector function for treatment of Alzheimer&#39;s disease and Aβ peptide associated diseases are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/754,579, filed Apr. 5, 2010, which is a divisionalapplication of U.S. patent application Ser. No. 11/194,989, filed Aug.1, 2005, now U.S. Pat. No. 7,927,594, issued on Apr. 19, 2011 and alsoclaims the priority benefit of U.S. provisional application Ser. Nos.60/592,494, filed Jul. 30, 2004; 60/653,197, filed Feb. 14, 2005; and60/676,093, filed Apr. 29, 2005; all of which are incorporated herein byreference in their entirety.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled “PC19496D_SeqListing_ST25.txt”created on Jul. 2, 2012 and having a size of 35 KB. The sequence listingcontained in this .txt file is part of the specification and is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention concerns antibodies to amyloid-beta peptide. The inventionfurther concerns use of such antibodies in the treatment and/orprevention of diseases, such as Alzheimer's disease.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a degenerative brain disorder characterizedclinically by progressive memory deficits, confusion, gradual physicaldeterioration and, ultimately, death. Approximately 15 million peopleworldwide are affected by Alzheimer's disease, and the number isexpected to increase dramatically as lifespan increases. Histologically,the disease is characterized by neuritic plaques, found primarily in theassociation cortex, limbic system and basal ganglia. The majorconstituent of these plaques is amyloid beta peptide (Aβ), which is thecleavage product of beta amyloid precursor protein (βAPP or APP). APP isa type I transmembrane glycoprotein that contains a large ectopicN-terminal domain, a transmembrane domain, and a small cytoplasmicC-terminal tail. Alternative splicing of the transcript of the singleAPP gene on chromosome 21 results in several isoforms that differ in thenumber of amino acids.

Aβ appears to have a central role in the neuropathology of Alzheimer'sdisease. Familial forms of the disease have been linked to mutations inAPP and the presenilin genes (Tanzi et al., 1996, Neurobiol. Dis.3:159-168; Hardy, 1996, Ann. Med. 28:255-258). Diseased-linked mutationsin these genes result in increased production of the 42-amino acid formof Aβ, the predominant form found in amyloid plaques. Moreover,immunization of transgenic mice that overexpress a disease-linked mutantform of APP with human Aβ reduces plaque burden and associatedpathologies (Schenk et al., 1999, Nature 400:173-177; WO 99/27944), andperipheral administration of antibodies directed against Aβ also reducesplaque burden in the brain (Bard et al., 2000, Nature Medicine6(8):916-919; WO 2004/032868; WO 00/72880).

It has been reported that Fc-mediated phagocytosis by microglial cellsand/or macrophages is important to the process of plaque clearance invivo. Bard et al., Proc. Natl. Acad. Sci. USA 100, 2023-2028 (2003).However, it has also been reported that non-Fc-mediated mechanisms areinvolved in clearance of amyloid-β in vivo by immunotherapy. Bacskai etal., J. Neurosci. 22:7873-7878 (2002); Das et al., J. Neurosci.23:8532-8538 (2003).

Antibody therapy therefore provides a promising approach to thetreatment and prevention of Alzheimer's disease. However, human clinicaltrials with a vaccine including Aβ1-42 were suspended due tomeningoencephalititis in a subset of patients. Orgogozo et al.,Neruology 61:7-8 (2003); Ferrer et al., Brain Pathol. 14:11-20 (2004).It has been reported that passive immunization with an N-terminalspecific anti-Aβ antibody results in a significant reduction of mainlydiffuse amyloid, but induces an increase of cerebral microhemorrhagefrequency in transgenic mice that exhibit the age-related development ofamyloid plaques and neurodegeneration as well as cerebral amyloidangiopathy (CAA) similar to that observed in the human AD brain. Pfeiferet al., Science 298:1379 (2002). It has been suggested that exacerbationof cerebral amyloid angiopathy (CAA)-associated microhemorrhage in APPtransgenic mice by passive immunization with antibody directed tobeta-amyloid is dependent on antibody recognition of deposited forms ofamyloid beta peptide. Racke et al., J. Neurosci. 25:629-636 (2005).Passive immunization with antibodies against a peptide component of anamyloid deposit, which antibodies are devoid of Fc regions, has beensuggested in order to decrease the risk of inflammation. WO 03/086310.There remains a need for antibodies and other immunotherapeutic agentsdirected against Aβ having improved efficacy and safety profile, andwhich are suitable for use with human patients.

Throughout this application various publications (including patents andpatent applications) are referenced. The disclosures of thesepublications in their entireties are hereby incorporated by reference.

BRIEF SUMMARY OF THE INVENTION Section I

The present invention provides methods for treating a diseasecharacterized by aberrant deposition of a protein in the brain of asubject. The methods comprise administering to the subject an effectiveamount of a pharmaceutical composition comprising an antibody thatspecifically binds to the protein or the protein deposit, or apolynucleotide encoding the antibody, wherein the antibody has impairedeffector function.

The invention also provides methods for treating or preventing diseasesassociated with amyloid deposit of Aβ (e.g., deposit in the brain tissueand cerebral vasculature) in a subject, such as Alzheimer's disease,Down's syndrome, multi-infarct dementia, mild cognitive impairment, andcerebral amyloid angiopathy. The method comprises administering to thesubject an effective amount of a pharmaceutical composition comprisingan antibody that specifically binds to a beta-amyloid peptide or anaggregated form of a beta-amyloid peptide, or a polynucleotide encodingthe antibody, wherein the antibody has impaired effector function.

The invention also provides methods of delaying development of a symptomassociated with diseases associated with amyloid deposit of Aβ in asubject, such as Alzheimer's disease, comprising administering to thesubject an effective dosage of a pharmaceutical composition comprisingan antibody that specifically binds to a beta-amyloid peptide or anaggregated form of a beta-amyloid peptide, or a polynucleotide encodingthe antibody, wherein the antibody has impaired effector function.

The invention also provides methods of suppressing formation of amyloidplaques and/or amyloid accumulation in a subject comprisingadministering to the subject an effective dosage of a pharmaceuticalcomposition comprising an antibody that specifically binds to abeta-amyloid peptide or an aggregated form of a beta-amyloid peptide, ora polynucleotide encoding the antibody, wherein the antibody hasimpaired effector function. In some embodiments, the amyloid plaques arein the brain (brain tissue) of the subject. In some embodiments, theamyloid plaques are in the cerebral vasculature. In some embodiments,the amyloid accumulation is in the circulatory system.

The invention also provides methods of reducing amyloid plaques and/oramyloid accumulation in a subject comprising administering to thesubject an effective dosage of a pharmaceutical composition comprisingan antibody that specifically binds to a beta-amyloid peptide or anaggregated form of a beta-amyloid peptide, or a polynucleotide encodingthe antibody, wherein the antibody has impaired effector function. Insome embodiments, the amyloid plaques are in the brain (brain tissue) ofthe subject. In some embodiments, the amyloid plaques are in thecerebral vasculature. In some embodiments, the amyloid accumulation isin the circulatory system.

The invention also provides methods of removing or clearing amyloidplaques and/or amyloid accumulation in a subject comprisingadministering to the subject an effective dosage of a pharmaceuticalcomposition comprising an antibody that specifically binds to abeta-amyloid peptide or an aggregated form of a beta-amyloid peptide, ora polynucleotide encoding the antibody, wherein the antibody hasimpaired effector function. In some embodiments, the amyloid plaques arein the brain (brain tissue) of the subject. In some embodiments, theamyloid plaques are in the cerebral vasculature. In some embodiments,the amyloid accumulation is in the circulatory system.

The invention also provides methods for inhibiting the accumulation ofAβ peptide in a tissue comprising contacting the tissue with an antibodythat specifically binds to a beta-amyloid peptide or an aggregated formof a beta-amyloid peptide, wherein the antibody has impaired effectorfunction.

The invention also provides methods of reducing Aβ peptide (such assoluble, oligomeric, and deposited form) in a subject comprisingadministrating to the subject an effective amount of an antibody thatspecifically binds to a beta-amyloid peptide or an aggregated form of abeta-amyloid peptide, or a polynucleotide encoding the antibody, whereinthe antibody has impaired effector function. In some embodiments, theaccumulation of Aβ peptide is inhibited and/or reduced in the brain. Insome embodiments, the toxic effects of Aβ peptide are inhibited and/orreduced. Thus, the method of the invention can be used to treat anydisease in which accumulation of Aβ peptide is present or suspected,such as Alzheimer's disease, Down's syndrome, Parkinson's disease, andmulti-infarct dementia.

The invention also provides methods of improving cognition or reversingcognitive decline associated with diseases associated with amyloiddeposit of Aβ in a subject, such as Alzheimer's disease, comprisingadministering to the subject an effective dosage of a pharmaceuticalcomposition comprising an antibody that specifically binds to abeta-amyloid peptide or an aggregated form of a beta-amyloid peptide, ora polynucleotide encoding the antibody, wherein the antibody hasimpaired effector function.

The invention also provides methods for treating or preventing diseasesassociated with amyloid deposit of Aβ, comprising administering to thesubject an effective dosage of a pharmaceutical composition comprisingan antibody that specifically binds to a beta-amyloid peptide or anaggregated form of a beta-amyloid peptide, wherein the antibodycomprises an Fc region with a variation from a naturally occurring Fcregion, wherein the variation results in impaired effector function. Insome embodiments, the administration of the antibody causes lesscerebral microhemorrhage than administration of an antibody without thevariation.

Polypeptides that specifically bind to an Aβ peptide or an aggregatedform of an Aβ peptide and comprises a heavy chain constant region havingimpaired effector function may also be used for any of the methodsdescribed herein. In some embodiments, the polypeptide comprises asequence (e.g., one or more CDRs) derived from antibody 9TL or itsvariants shown in Table 3. In some embodiments, the polypeptidecomprises a sequence (e.g., one or more CDRs) derived from antibody 6G.

The antibody and polypeptide used for the methods of the inventionspecifically bind to an Aβ peptide or an aggregated form of an Aβpeptide, but have impaired effector function. In some embodiments, theantibody or polypeptide is not a F(ab′)₂ fragment. In some embodiments,the antibody or polypeptide is not a Fab fragment. In some embodiments,the antibody or polypeptide is not a single chain antibody scFv.

In some embodiments, the antibody or the polypeptide comprises a heavychain constant region having impaired effector function, wherein theheavy chain constant region comprises an Fc region. In some embodiments,the N-glycosylation in the Fc region is removed. In some embodiments,the Fc region comprises a mutation within the N-glycosylationrecognition sequence, whereby the Fc region of the antibody orpolypeptide is not N-glycosylated. In some embodiments, the Fc region isPEGylated. In some embodiments, the heavy chain constant region of theantibody or the polypeptide is a human heavy chain IgG2a constant regioncontaining the following mutations: A330P331 to S330S331 (amino acidnumbering with reference to the wildtype IgG2a sequence). In someembodiments, the antibody or the polypeptide comprises a constant regionof IgG4 comprising the following mutations: E233F234L235 toP233V234A235.

In some embodiments, the antibody or polypeptide specifically binds toan epitope within residues 1-16 of Aβ peptide. In some embodiments, theantibody or polypeptide specifically binds to the N-terminus of the Aβpeptide. In some embodiments, the antibody or the polypeptidespecifically binds to an epitope within residues 16-28 of Aβ peptide. Insome embodiments, the antibody specifically binds to an epitope on theC-terminal side of an Aβ peptide, such as an epitope starting from aminoacid 25 or later. The antibody may specifically bind to the freeC-terminus amino acid of C-terminus truncated Aβ peptide, for example,Aβ 1-37, 1-38, 1-39, 1-40, 1-41, 1-42, 1-43. In some embodiments, theantibody or the polypeptide specifically binds to an epitope withinresidues 28-40 of Aβ₁₋₄₀ peptide. In some embodiments, the antibody orthe polypeptide specifically binds to an epitope within residues 28-42of Aβ₁₋₄₂ peptide. In some embodiments, the antibody or the polypeptidespecifically binds to an epitope within residues 28-43 of Aβ₁₋₄₃peptide. In some embodiments, the antibody or the polypeptidespecifically binds to Aβ peptide without binding to full-length amyloidprecursor protein (APP). In some embodiments, the antibody or thepolypeptide specifically binds to the aggregated form of Aβ withoutbinding to the soluble form. In some embodiments, the antibody or thepolypeptide specifically binds to the soluble form of Aβ without bindingto the aggregated form. In some embodiments, the antibody or thepolypeptide specifically binds to both aggregated form and soluble formsof Aβ.

In some embodiments, the antibody or the polypeptide specifically bindsto a C-terminal peptide 33-40 of Aβ₁₋₄₀. In some embodiments, theantibody or the polypeptide specifically binds to an epitope on Aβ₁₋₄₀that includes amino acid 35-40. In some embodiments, the antibody or thepolypeptide specifically binds to an epitope on Aβ₁₋₄₀ that includesamino acid 36-40. In some embodiments, the antibody or the polypeptidespecifically binds to an epitope on Aβ₁₋₄₀ that includes amino acid 39and/or 40. In some embodiments, the antibody or the polypeptidespecifically binds to Aβ₁₋₄₀ but do not specifically bind to Aβ₁₋₄₂and/or Aβ₁₋₄₃. In some embodiments, the antibody comprises the variableregion of antibody 9TL or an antibody derived from 9TL described herein.In some embodiments, the antibody or polypeptide competitively inhibitsbinding of antibody 9TL and/or antibody or polypeptide derived from 9TLto Aβ₁₋₄₀.

In some embodiments, the antibody or the polypeptide binds to Aβ₁₋₄₀with higher affinity than its binding to Aβ₁₋₄₂ and Aβ₁₋₄₃. In someembodiments, the antibody binds to an epitope on Aβ₁₋₄₀ that includesamino acids 25-34 and 40. In some embodiments, the antibody comprisesthe variable region of antibody 6G or an antibody derived from 6Gdescribed herein. In some embodiments, the antibody or polypeptidecompetitively inhibits binding of antibody 6G and/or antibody orpolypeptide derived from 6G to Aβ.

Administration of antibody or polypeptide that specifically binds to anAβ peptide and has impaired effector function may be by any means knownin the art, including: intravenously, subcutaneously, via inhalation,intraarterially, intramuscularly, intracardially, intraventricularly,parenteral, intrathecally, and intraperitoneally. Administration may besystemic, e.g. intravenously, or localized. This also generally appliesto polypeptides and polynucleotides of the invention.

The invention also provides pharmaceutical composition comprising aneffective amount of any of the antibodies or polypeptides thatspecifically bind to an Aβ peptide or an aggregated form of an Aβpeptide and have impaired effector function, or polynucleotides encodingthe antibodies or polypeptides, and a pharmaceutical acceptableexcipient.

The invention also provides kits and compositions comprising any one ormore of the compositions comprising an effective amount of any of theantibodies or polypeptides that specifically bind to an Aβ peptide or anaggregated form of an Aβ peptide and have impaired effector function, orpolynucleotides encoding the antibodies or polypeptides. These kits,generally in suitable packaging and provided with appropriateinstructions, are useful for any of the methods described herein.

The invention also provides a method of producing a therapeutichumanized antibody for treatment of a disease associated with amyloiddeposits of Aβ peptide in the brain of a human subject, comprisingselecting a first humanized antibody that specifically binds to Aβpeptide; and altering the Fc region of the antibody to provide atherapeutic humanized antibody having impaired effector functionrelative to the first humanized antibody.

Section II

The invention disclosed herein concerns antibodies that bind toC-terminus of Aβ₁₋₄₀ peptide (SEQ ID NO:15 shown in Table 4).Accordingly, in one aspect, the invention is an antibody 9TL(interchangeably termed “9TL”) that is produced by expression vectorshaving ATCC Accession Nos. PTA-6124 and PTA-6125. The amino acidsequences of the heavy chain and light chain variable regions of 9TL areshown in FIG. 1. The complementarity determining region (CDR) portionsof antibody 9TL (including Chothia and Kabat CDRs) are also shown inFIG. 1. It is understood that reference to any part of or entire regionof 9TL encompasses sequences produced by the expression vectors havingATCC Accession Nos. PTA-6124 and PTA-6125, and/or the sequences depictedin FIG. 1.

In another aspect, the invention also provides antibody variants of 9TLwith amino acid sequences depicted in Table 3.

In another aspect, the invention is an antibody comprising a fragment ora region of the antibody 9TL or its variants shown in Table 3. In oneembodiment, the fragment is a light chain of the antibody 9TL. Inanother embodiment, the fragment is a heavy chain of the antibody 9TL.In yet another embodiment, the fragment contains one or more variableregions from a light chain and/or a heavy chain of the antibody 9TL. Inyet another embodiment, the fragment contains one or more variableregions from a light chain and/or a heavy chain shown in FIG. 1. In yetanother embodiment, the fragment contains one or more CDRs from a lightchain and/or a heavy chain of the antibody 9TL.

In another aspect, the invention provides polypeptides (which may or maynot be an antibody) comprising any one or more of the following: a) oneor more CDR(s) of antibody 9TL or its variants shown in Table 3; b) CDRH3 from the heavy chain of antibody 9TL or its variants shown in Table3; c) CDR L3 from the light chain of antibody 9TL or its variants shownin Table 3; d) three CDRs from the light chain of antibody 9TL or itsvariants shown in Table 3; e) three CDRs from the heavy chain ofantibody 9TL or its variants shown in Table 3; f) three CDRs from thelight chain and three CDRs from the heavy chain of antibody 9TL or itsvariants shown in Table 3. The invention further provides polypeptides(which may or may not be an antibody) comprising any one or more of thefollowing: a) one or more (one, two, three, four, five, or six) CDR(s)derived from antibody 9TL or its variants shown in Table 3; b) a CDRderived from CDR H3 from the heavy chain of antibody 9TL; and/or c) aCDR derived from CDR L3 from the light chain of antibody 9TL. In someembodiments, the CDR is a CDR shown in FIG. 1. In some embodiments, theone or more CDRs derived from antibody 9TL or its variants shown inTable 3 are at least about 85%, at least about 86%, at least about 87%,at least about 88%, at least about 89%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99% identical to at least one, at leasttwo, at least three, at least four, at least five, or at least six CDRsof 9TL or its variants.

In some embodiments, the CDR is a Kabat CDR. In other embodiments, theCDR is a Chothia CDR. In other embodiments, the CDR is a combination ofa Kabat and a Chothia CDR (also termed “combined CDR” or “extendedCDR”). In other words, for any given embodiment containing more than oneCDR, the CDRs may be any of Kabat, Chothia, and/or combined.

In some embodiments, the polypeptide (such as an antibody) comprises anamino acid sequence shown in SEQ ID NO:5, wherein L1 is L, V, or I;wherein Y2 is Y or W; wherein S3 is S, T, or G; wherein L4 is L, R, A,V, S, T, Q, or E; wherein V6 is V, I, T, P, C, Q, S, N, or F; andwherein Y7 is Y, H, F, W, S, I, V, or A. In some embodiments, the aminoacid sequence is a CDR3 in a heavy chain variable region. Forconvenience herein, “is” in this context or reference to an amino acidrefers to choices of amino acid(s) for a given position with referenceto the position in the SEQ ID. For example, “L1 is L, V, or I” refers toamino acid L at position 1 in SEQ ID NO:5 may be substituted with V orI.

In some embodiments, the polypeptide (such as an antibody) comprises anamino acid sequence shown in SEQ ID NO:6, wherein Y8 is Y, A, or H; andwherein A11 is A or S; and wherein K12 is K or A. In some embodiments,the amino acid sequence is a CDR1 in a light chain variable region.

In some embodiments, the polypeptide (such as an antibody) comprises anamino acid sequence shown in SEQ ID NO:8, wherein L1 is L, M, N, C, F,V, K, S, Q, G, S; wherein G3 is G, S, or T; wherein T4 is T or S;wherein H5 is H or L; wherein Y6 is Y, P, A, W, Q, M, S, or E; whereinV8 is V, L, K, H, T, A, E, or M; and wherein L9 is L, I, T, S, or V. Insome embodiments, the amino acid sequence is a CDR3 in a light chainvariable region.

In some embodiments, the polypeptide (such as an antibody) comprises aheavy chain variable region comprising (a) a CDR1 region shown in SEQ IDNO:3; (b) a CDR2 region shown in SEQ ID NO:4; and (c) a CDR3 regionshown in SEQ ID NO:5, wherein L1 is L, V, or I; wherein Y2 is Y or W;wherein S3 is S, T, or G; wherein L4 is L, R, A, V, S, T, Q, or E;wherein V6 is V, I, T, P, C, Q, S, N, or F; and wherein Y7 is Y, H, F,W, S, I, V, or A.

In some embodiments, the polypeptide (such as an antibody) comprises alight chain variable region comprising (a) a CDR1 region shown in SEQ IDNO:6, wherein Y8 is Y, A, or H; and wherein A11 is A or S; and whereinK12 is K or A; (b) a CDR2 region shown in SEQ ID NO:7; and (c) a CDR3region shown in SEQ ID NO:8, wherein L1 is L, M, N, C, F, V, K, S, Q, G,S; wherein G3 is G, S, or T; wherein T4 is T or S; wherein H5 is H or L;wherein Y6 is Y, P, A, W, Q, M, S, or E; wherein V8 is V, L, K, H, T, A,E, or M; and wherein L9 is L, I, T, S, or V.

In some embodiments, the antibody of the invention is a human antibody.In other embodiments, the antibody of the invention is a humanizedantibody. In some embodiments, the antibody is monoclonal. In someembodiments, the antibody (or polypeptide) is isolated. In someembodiments, the antibody (or polypeptide) is substantially pure.

The heavy chain constant region of the antibodies may be from any typesof constant region, such as IgG, IgM, IgD, IgA, and IgE; and anyisotypes, such as IgG1, IgG2, IgG3, and IgG4.

In some embodiments, the antibody comprises a modified constant region,such as a constant region that is immunologically inert (which includespartially immunologically inert, and is used interchangeably with theterm “having impaired effector function”), e.g., does not triggercomplement mediated lysis, does not stimulate antibody-dependent cellmediated cytotoxicity (ADCC), or does not activate microglia. In someembodiments, the constant region is modified as described in Eur. J.Immunol. (1999) 29:2613-2624; PCT Application No. PCT/GB99/01441; and/orUK Patent Application No. 9809951.8. In other embodiments, the antibodycomprises a human heavy chain IgG2a constant region comprising thefollowing mutations: A330P331 to S330S331 (amino acid numbering withreference to the wildtype IgG2a sequence). Eur. J. Immunol. (1999)29:2613-2624. In some embodiments, the antibody comprises a constantregion of IgG4 comprising the following mutations: E233F234L235 toP233V234A235. In still other embodiments, the constant region isaglycosylated for N-linked glycosylation. In some embodiments, theconstant region is aglycosylated for N-linked glycosylation by mutatingthe oligosaccharide attachment residue (such as Asn297) and/or flankingresidues that are part of the N-glycosylation recognition sequence inthe constant region. In some embodiments, the constant region isaglycosylated for N-linked glycosylation. The constant region may beaglycosylated for N-linked glycosylation enzymatically or by expressionin a glycosylation deficient host cell.

In another aspect, the invention provides a polynucleotide (which may beisolated) comprising a polynucleotide encoding a fragment or a region ofthe antibody 9TL or its variants shown in Table 3. In one embodiment,the fragment is a light chain of the antibody 9TL. In anotherembodiment, the fragment is a heavy chain of the antibody 9TL. In yetanother embodiment, the fragment contains one or more variable regionsfrom a light chain and/or a heavy chain of the antibody 9TL. In yetanother embodiment, the fragment contains one or more (i.e., one, two,three, four, five, six) complementarity determining regions (CDRs) froma light chain and/or a heavy chain of the antibody 9TL.

In another aspect, the invention is a polynucleotide (which may beisolated) comprising a polynucleotide that encodes for antibody 9TL orits variants shown in Table 3. In some embodiments, the polynucleotidecomprises either or both of the polynucleotides shown in SEQ ID NO:9 andSEQ ID NO:10.

In another aspect, the invention provides polynucleotides encoding anyof the antibodies (including antibody fragments) or polypeptidesdescribed herein.

In another aspect, the invention provides vectors (including expressionand cloning vectors) and host cells comprising any of the polynucleotidedisclosed herein. In some embodiments, the vector is pDb.9TL.hFc2ahaving ATCC No. PTA-6124. In other embodiments, the vector is pEb.9TL.hKhaving ATCC No. PTA-6125.

In another aspect, the invention is a host cell comprising apolynucleotide encoding any of the antibodies described herein.

In another aspect, the invention is a complex of Aβ₁₋₄₀ bound byantibody 9TL or its variants shown in Table 3.

In another aspect, the invention is a complex of Aβ₁₋₄₀ bound by any ofthe antibodies or polypeptides described herein.

In another aspect, the invention is a pharmaceutical compositioncomprising an effective amount of any of the polypeptides (includingantibodies, such as an antibody comprising one or more CDRs of antibody9TL) or polynucleotides described herein, and a pharmaceuticallyacceptable excipient.

In another aspect, the invention is a method of generating antibody 9TLcomprising culturing a host cell or progeny thereof under conditionsthat allow production of antibody 9TL, wherein the host cell comprisesan expression vector that encodes for antibody 9TL; and, in someembodiments, purifying the antibody 9TL. In some embodiments, theexpression vector comprises one or both of the polynucleotide sequencesshown in SEQ ID NO:9 and SEQ ID NO:10.

In another aspect, the invention provides methods of generating any ofthe antibodies or polypeptides described herein by expressing one ormore polynucleotides encoding the antibody (which may be separatelyexpressed as a single light or heavy chain, or both a light and a heavychain are expressed from one vector) or the polypeptide in a suitablecell, generally followed by recovering and/or isolating the antibody orpolypeptides of interest.

The invention also provides a method for preventing, treating,inhibiting, or delaying the development of Alzheimer's disease and otherdiseases associated with altered Aβ or βAPP expression, or accumulationof Aβ peptide, such as Down's syndrome, Parkinson's disease,multi-infarct dementia, mild cognitive impairment, cerebral amyloidangiopathy, and AIDS. The method comprises administering an effectivedosage a pharmaceutical composition comprising an antibody, apolypeptide, or a polynucleotide of the invention to a subject.

The invention also provides a method of delaying development of asymptom associated with Alzheimer's disease or other diseases related toaccumulation of Aβ peptide in a subject comprising administering aneffective dosage of a pharmaceutical composition comprising an antibody,a polypeptide, or a polynucleotide of the invention to the subject.

The invention also provides a method of suppressing formation of amyloidplaques and/or amyloid accumulation in a subject comprisingadministering an effective dosage of a pharmaceutical compositioncomprising an antibody, a polypeptide, or a polynucleotide of theinvention to the subject. In some embodiments, the amyloid plaques arein the brain (brain tissue) of the subject. In some embodiments, theamyloid plaques are in the cerebral vasculature. In other embodiments,the amyloid accumulation is in the circulatory system.

The invention also provides a method of reducing amyloid plaques and/oramyloid accumulation in a subject comprising administering an effectivedosage of a pharmaceutical composition comprising an antibody, apolypeptide, or a polynucleotide of the invention to the subject. Insome embodiments, the amyloid plaques are in the brain (brain tissue) ofthe subject. In some embodiments, the amyloid plaques are in thecerebral vasculature. In other embodiments, the amyloid accumulation isin the circulatory system.

The invention also provides a method of removing or clearing amyloidplaques and/or amyloid accumulation in a subject comprisingadministering an effective dosage of a pharmaceutical compositioncomprising an antibody, a polypeptide, or a polynucleotide of theinvention to the subject. In some embodiments, the amyloid plaques arein the brain (brain tissue) of the subject. In some embodiments, theamyloid plaques are in the cerebral vasculature. In other embodiments,the amyloid accumulation is in the circulatory system.

Additionally, the invention provides a method for inhibiting theaccumulation of Aβ peptide in a tissue comprising contacting the tissuewith an antibody or a polypeptide of the invention.

The invention also provides a method of reducing Aβ peptide (such assoluble, oligomeric and deposited form) in the brain of an individualcomprising administering to the individual an effective amount of anantibody or a polypeptide of the invention. In some embodiments, theaccumulation of Aβ peptide is inhibited and/or reduced in the brain. Insome embodiments, the toxic effects of Aβ peptide are inhibited and/orreduced. Thus, the method of the invention can be used to treat anydisease in which accumulation of Aβ peptide is present or suspected,such as Alzheimer's disease, Down's syndrome, Parkinson's disease,multi-infarct dementia, mild cognitive impairment, and cerebral amyloidangiopathy.

The invention also provides methods of improving cognition or reversingcognitive decline associated with diseases associated with amyloiddeposit of Aβ in the brain of an individual, such as Alzheimer'sdisease, comprising administering an effective dosage of apharmaceutical composition comprising an antibody, a polypeptide, or apolynucleotide of the invention to the individual.

Any antibodies, polypeptides, or polynucleotides described herein may beused for the methods of the invention. In some embodiments, the antibodyis antibody 9TL.

Antibodies and polypeptides of the invention can further be used in thedetection, diagnosis and monitoring of Alzheimer's disease and otherdiseases associated with altered Aβ or βAPP expression, such as Down'ssyndrome, and AIDS. The method comprises contacting a specimen of apatient suspected of having altered Aβ or βAPP expression with anantibody of the invention and determining whether the level of Aβ orβAPP differs from that of a control or comparison specimen. In someembodiments, serum level of Aβ is measured before and afteradministration of an anti-Aβ antibody; and any increase of serum levelof Aβ is assessed.

Administration of any antibody or polypeptide of the invention may be byany means known in the art, including: intravenously, subcutaneously,via inhalation, intraarterially, intramuscularly, intracardially,intraventricularly, parenteral, intrathecally, and intraperitoneally.Administration may be systemic, e.g. intravenously, or localized. Thisalso generally applies to polypeptides and polynucleotides of theinvention.

In another aspect, the invention provides kits and compositionscomprising any one or more of the compositions described herein. Thesekits, generally in suitable packaging and provided with appropriateinstructions, are useful for any of the methods described herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 shows the amino acid sequence of the heavy chain variable region(SEQ ID NO:1) and light chain variable region (SEQ ID NO:2) of the 9TLantibody. The Kabat CDRs are in bold text, and the Chothia CDRs areunderlined. The amino acid residues for the heavy chain and light chainvariable region are numbered sequentially.

FIG. 2 shows epitope mapping of antibody 9TL by peptide competition.Aβ₁₋₄₀ peptide was immobilized on the SA chip. Monoclonal antibody 2289and 9TL Fab fragment (50 nM each), each of which was preincubated for 1h with 10 μM various peptide (amino acids 28-40, 1-40, 1-28, 28-42,22-35, 1-16, 1-43, 33-40, 1-38, or 17-40 of Aβ) or no peptide, and wasthen flowed onto the chip. Binding of the antibody Fab fragment toimmobilized Aβ₁₋₄₀ peptide was measured.

FIG. 3 is a graph showing epitope mapping of antibody 2H6 by peptidecompetition. Aβ₁₋₄₀ peptide was immobilized on the SA chip. Monoclonalantibody 2289, 2286, or 2H6 (100 nM each), each of which waspreincubated for 1 h with 16 μM various peptide (amino acids 1-16, 1-28,1-38, 1-40, 1-42, 1-43, 17-40, 17-42, 22-35, 25-35, or 33-40 of Aβ) orno peptide, was flowed onto the chip. Binding of the antibody toimmobilized Aβ₁₋₄₀ peptide was measured.

FIG. 4 is a graph showing binding of antibody 2H6, 2286, and 2289 todifferent Aβ peptide C-terminal variants. GST-Aβ variants (M35A, V36A,G37A, G38A, V39A, or V40A), or GST-Aβ peptide 1-39, 1-41, 1-40, 1-42were immobilized on ELISA plate. Monoclonal antibody 2286, 2H6, or 2289(0.3 nM each mAb) was incubated with each of the immobilized peptides,and their binding was detected by further incubating with biotinylatedanti-mouse IgG (H+L) and followed by Sterptavidin-HRP.

FIG. 5 is a graph showing spatial learning deficits in APP-transgenicmice were reversed following 16 weeks of antibody treatment with 2H6 anddeglycosylated 2H6. Mice were tested in a two-day version of theradial-arm water maze. Y axis represents mean number of errors made overthe 2-day trial period. Block numbers 1-5 represent tests in day 1; andblock numbers 6-10 represent tests in day 2. “*” indicates p<0.05 forboth 2H6 (A-2H6) and deglycosylated 2H6 (A-De-2H6) treated mice whencompared with anti-AMN antibody treated mice (A-AMN). “**” indicatesp<0.01 for both 2H6 (A-2H6) and deglycosylated 2H6 (A-De-2H6) treatedmice when compared with anti-AMN antibody treated mice (A-AMN).

FIGS. 6A and 6B are graphs showing decreases of parenchymal Congo-redstained amyloid-beta peptide in hippocampus (FIG. 6B) and frontal cortex(FIG. 6A) after 16 weeks of antibody treatment with 2H6, anti-AMN(referred to as AMN), and deglycosylated 2H6 (referred to as D-2H6)antibody. Y-axes in FIGS. 6A and 6B represent mean of percent areapositive for Congo-red staining X-axes in FIGS. 6A and 6B represent typeof antibody administered.

FIGS. 7A and 7B are graphs showing increases of vascular Congo-redstained amyloid-beta peptide in hippocampus (FIG. 7A) and frontal cortex(FIG. 7B) after 16 weeks of antibody treatment with 2H6, anti-AMN(referred to as AMN), and deglycosylated 2H6 (referred to as D-2H6)antibody. Y-axes in FIGS. 7A and 7B represent mean of percent areapositive for Congo-red staining X-axes in FIGS. 7A and 7B represent typeof antibody administered.

FIG. 8 is a graph showing number of Prussian blue positive profilesafter 16 weeks of antibody treatment with 2H6, anti-AMN (referred to asAMN), and deglycosylated 2H6 (referred to as D-2H6) antibody. Y-axisrepresents positive profiles per section. X-axis represents type ofantibody administered.

FIG. 9 is a graph showing serum level of Aβ peptide after administrationof anti-AMN antibody (referred to as AMN), antibody 2H6 (referred to as2H6), deglycosylated 2H6 (referred to as 2H6-D) in APP Tg2576 mice, andafter administration of anti-AMN antibody and antibody 2H6 in wild type(WT) mice.

FIG. 10 shows immunostaining of CD45 in the hippocampus of a mouse afterintracranial administration of 2H6 antibody (A) or deglycosylated 2H6antibody (B). The bottom panel shows that the ratio of the average areaoccupied CD45 positive staining of injected side over uninjected side inthe frontal cortex and hippocampus after intracranial administration ofthe control antibody, 2H6 antibody, or deglycosylated 2H6 antibody. “*”indicates P<0.01 as compared to the control antibody.

FIG. 11 shows immunostaining of Fcγ receptor in the hippocampus of amouse after intracranial administration of 2H6 antibody (A) ordeglycosylated 2H6 antibody (B). The bottom panel shows that the ratioof the average area occupied by Fcγ receptor positive staining ofinjected side over uninjected side in the frontal cortex and hippocampusafter intracranial administration of the control antibody, 2H6 antibody,or deglycosylated 2H6 antibody. “**” indicates P<0.01 as compared to thecontrol antibody.

FIG. 12 shows immunostaining of Aβ peptide in the hippocampus of a mouseafter intracranial administration of 2H6 antibody (A) or deglycosylated2H6 antibody (B). The bottom panel shows that the ratio of the averagearea occupied by Aβ positive staining of injected side over uninjectedside in the frontal cortex and hippocampus after intracranialadministration of the control antibody, 2H6 antibody, or deglycosylated2H6 antibody. “*” indicates P<0.01 as compared to the control antibody.“*” indicates P<0.05 as compared to the control antibody.

FIG. 13 shows thioflavine-S in the hippocampus of a mouse afterintracranial administration of 2H6 antibody (A) or deglycosylated 2H6antibody (B). The bottom panel shows that the ratio of the average areaoccupied by thioflavine-S positive staining of injected side overuninjected side in the frontal cortex and hippocampus after intracranialadministration of the control antibody, 2H6 antibody, or deglycosylated2H6 antibody. “*” indicates P<0.05 as compared to the control antibody.

FIG. 14 shows epitope mapping of antibody 2294 and 6G by ELISA. VariousAβ peptides were immobilized on ELISA plates. Antibodies were incubatedfor 1 hour with various immobilized peptides. Antibody 6G bound toimmobilized Aβ peptides were measured using goat anti-human kappa HRPconjugated secondary antibody. Antibody 2294 bound to immobilized Aβpeptides were measured using goat anti-mouse that binds to both heavyand light chain and is HRP conjugated secondary antibody. “NB” refers tono binding detected. The numbers in the columns under “2294” and “6G”represent absorbance at 450 nm. Aβ peptides from the top to the bottomare assigned SEQ ID NOS:41-53, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The invention disclosed herein provides antibodies and polypeptides thatbind to C-terminus of Aβ₁₋₄₀. These antibodies and polypeptides arederived from 9TL or its variants shown in Table 3. The invention alsoprovides methods of making and using these antibodies. In someembodiments, the invention provides antibody 9TL, and methods of makingand using this antibody. The invention also provides 9TL polypeptides(including antibodies) that bind Aβ₁₋₄₀, and polynucleotides encoding9TL antibody and/or polypeptide.

The invention disclosed herein also provides methods for preventingand/or treating Aβ-associated diseases, such as Alzheimer's disease,Down's syndrome, Parkinson's disease, multi-infarct dementia, mildcognitive impairment, cerebral amyloid angiopathy, vascular disordercaused by deposit of Aβ peptide in blood vessels (such as stroke andHCHWA-D) in an individual by administration of a therapeuticallyeffective amount of an antibody 9TL, or antibody or polypeptide derivedfrom 9TL.

The invention also provides methods for treating or preventing diseasesassociated with β-amyloid deposit in an individual, such as Alzheimer'sdisease, Down's syndrome, multi-infarct dementia, mild cognitiveimpairment, and cerebral amyloid angiopathy in an individual byadministering to the individual an effective amount of a pharmaceuticalcomposition comprising an antibody or a polypeptide that specificallybinds to a β-amyloid peptide or an aggregated form of an Aβ peptide, ora polynucleotide encoding the antibody or the polypeptide, wherein theantibody or the polypeptide has impaired effector function.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (AcademicPress, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

DEFINITIONS

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain(ScFv), mutants thereof, fusion proteins comprising an antibody portion,and any other modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site. An antibody includes an antibodyof any class, such as IgG, IgA, or IgM (or sub-class thereof), and theantibody need not be of any particular class. Depending on the antibodyamino acid sequence of the constant domain of its heavy chains,immunoglobulins can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, 1975, Nature, 256:495, ormay be made by recombinant DNA methods such as described in U.S. Pat.No. 4,816,567. The monoclonal antibodies may also be isolated from phagelibraries generated using the techniques described in McCafferty et al.,1990, Nature, 348:552-554, for example.

As used herein, “humanized” antibodies refer to forms of non-human (e.g.murine) antibodies that are specific chimeric immunoglobulins,immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) thatcontain minimal sequence derived from non-human immunoglobulin. For themost part, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a complementary determining region(CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat, or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Antibodies mayhave Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,six) which are altered with respect to the original antibody, which arealso termed one or more CDRs “derived from” one or more CDRs from theoriginal antibody.

As used herein, “human antibody” means an antibody having an amino acidsequence corresponding to that of an antibody produced by a human and/orhas been made using any of the techniques for making human antibodiesknown in the art or disclosed herein. This definition of a humanantibody includes antibodies comprising at least one human heavy chainpolypeptide or at least one human light chain polypeptide. One suchexample is an antibody comprising murine light chain and human heavychain polypeptides. Human antibodies can be produced using varioustechniques known in the art. In one embodiment, the human antibody isselected from a phage library, where that phage library expresses humanantibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314;Sheets et al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter,1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol.,222:581). Human antibodies can also be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. This approach is described in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.Alternatively, the human antibody may be prepared by immortalizing humanB lymphocytes that produce an antibody directed against a target antigen(such B lymphocytes may be recovered from an individual or may have beenimmunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J.Immunol., 147 (1):86-95; and U.S. Pat. No. 5,750,373.

As used herein, the terms “9TL” and “antibody 9TL” are usedinterchangeably to refer to an antibody produced by expression vectorshaving deposit numbers of ATCC PTA-6124 and ATCC PTA-6125. The aminoacid sequence of the heavy chain and light chain variable regions areshown in FIG. 1. The CDR portions of antibody 9TL (including Chothia andKabat CDRs) are diagrammatically depicted in FIG. 1. The polynucleotidesencoding the heavy and light chain variable regions are shown in SEQ IDNO:9 and SEQ ID NO:10. The characterization of 9TL is described in theExamples.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon an antibody, the polypeptides can occur as single chains orassociated chains.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidemay comprise modified nucleotides, such as methylated nucleotides andtheir analogs. If present, modification to the nucleotide structure maybe imparted before or after assembly of the polymer. The sequence ofnucleotides may be interrupted by non-nucleotide components. Apolynucleotide may be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsinclude, for example, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.)and with charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.), those containing pendant moieties, such as, for example, proteins(e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine,etc.), those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent in the sugars may be replaced, for example, by phosphonategroups, phosphate groups, protected by standard protecting groups, oractivated to prepare additional linkages to additional nucleotides, ormay be conjugated to solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupmoieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′—O-methyl-,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,a-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages may be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S(“thioate”), P(S)S(“dithioate”), “(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Al-lazikani et al (1997) J.Molec. Biol. 273:927-948)). As used herein, a CDR may refer to CDRsdefined by either approach or by a combination of both approaches.

A “constant region” of an antibody refers to the constant region of theantibody light chain or the constant region of the antibody heavy chain,either alone or in combination.

An epitope that “preferentially binds” or “specifically binds” (usedinterchangeably herein) to an antibody or a polypeptide is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecule is saidto exhibit “specific binding” or “preferential binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular cell or substance than it doeswith alternative cells or substances. An antibody “specifically binds”or “preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to an Aβ₁₋₄₀ epitope is an antibody that binds thisepitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other Aβ₁₋₄₀ epitopes or non-Aβ₁₋₄₀epitopes. It is also understood by reading this definition that, forexample, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), more preferably at least90% pure, more preferably at least 95% pure, more preferably at least98% pure, more preferably at least 99% pure.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

The term “Fc region” is used to define a C-terminal region of animmunoglobulin heavy chain. The “Fc region” may be a native sequence Fcregion or a variant Fc region. Although the boundaries of the Fc regionof an immunoglobulin heavy chain might vary, the human IgG heavy chainFc region is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. Thenumbering of the residues in the Fc region is that of the EU index as inKabat. Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md., 1991. The Fc region of an immunoglobulin generally comprises twoconstant domains, CH2 and CH3.

As used herein, “Fc receptor” and “FcR” describe a receptor that bindsto the Fc region of an antibody. The preferred FcR is a native sequencehuman FcR. Moreover, a preferred FcR is one which binds an IgG antibody(a gamma receptor) and includes receptors of the FcγRI, FcγRII, andFcγRIII subclasses, including allelic variants and alternatively splicedforms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. FcRs are reviewed in Ravetch and Kinet,1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods,4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. “FcR”also includes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., 1976, J. Immunol.,117:587; and Kim et al., 1994, J. Immunol., 24:249).

“Complement dependent cytotoxicity” and “CDC” refer to the lysing of atarget in the presence of complement. The complement activation pathwayis initiated by the binding of the first component of the complementsystem (Clq) to a molecule (e.g. an antibody) complexed with a cognateantigen. To assess complement activation, a CDC assay, e.g. as describedin Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996), may beperformed.

A “functional Fc region” possesses at least one effector function of anative sequence Fc region. Exemplary “effector functions” include Clqbinding; complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;down-regulation of cell surface receptors (e.g. B cell receptor; BCR),etc. Such effector functions generally require the Fc region to becombined with a binding domain (e.g. an antibody variable domain) andcan be assessed using various assays known in the art for evaluatingsuch antibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification, yet retains at least one effector function of the nativesequence Fc region. Preferably, the variant Fc region has at least oneamino acid substitution compared to a native sequence Fc region or tothe Fc region of a parent polypeptide, e.g. from about one to about tenamino acid substitutions, and preferably from about one to about fiveamino acid substitutions in a native sequence Fc region or in the Fcregion of the parent polypeptide. The variant Fc region herein willpreferably possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, andmost preferably at least about 90% sequence identity therewith, morepreferably at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99% sequence identity therewith.

As used herein “antibody-dependent cell-mediated cytotoxicity” and“ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxiccells that express Fc receptors (FcRs) (e.g. natural killer (NK) cells,neutrophils, and macrophages) recognize bound antibody on a target celland subsequently cause lysis of the target cell. ADCC activity of amolecule of interest can be assessed using an in vitro ADCC assay, suchas that described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and NK cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al., 1998, PNAS (USA),95:652-656.

As used herein, an “effective dosage” or “effective amount” drug,compound, or pharmaceutical composition is an amount sufficient toeffect beneficial or desired results. For prophylactic use, beneficialor desired results include results such as eliminating or reducing therisk, lessening the severity, or delaying the outset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such asinhibiting, suppressing or reducing the formation of amyloid plaques,reducing, removing, clearing amyloid plaques, improving cognition,reversing or slowing cognitive decline, sequestering or increasingsoluble Aβ peptide circulating in biological fluids, decreasing one ormore symptoms resulting from the disease (biochemical, histologicaland/or behavioral), including its complications and intermediatepathological phenotypes presenting during development of the disease,increasing the quality of life of those suffering from the disease,decreasing the dose of other medications required to treat the disease,enhancing effect of another medication, delaying the progression of thedisease, and/or prolonging survival of patients. An effective dosage canbe administered in one or more administrations. For purposes of thisinvention, an effective dosage of drug, compound, or pharmaceuticalcomposition is an amount sufficient to accomplish prophylactic ortherapeutic treatment either directly or indirectly. As is understood inthe clinical context, an effective dosage of a drug, compound, orpharmaceutical composition may or may not be achieved in conjunctionwith another drug, compound, or pharmaceutical composition. Thus, an“effective dosage” may be considered in the context of administering oneor more therapeutic agents, and a single agent may be considered to begiven in an effective amount if, in conjunction with one or more otheragents, a desirable result may be or is achieved.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: inhibiting,suppressing or reducing the formation of amyloid plaques, reducing,removing, or clearing amyloid plaques, improving cognition, reversing orslowing cognitive decline, sequestering soluble Aβ peptide circulatingin biological fluids, reducing Aβ peptide (including soluble, oligomericand deposited) in a tissue (such as brain), inhibiting, slowing and/orreducing accumulation of Aβ peptide in the brain, inhibiting, slowingand/or reducing toxic effects of Aβ peptide in a tissue (such as brain),decreasing symptoms resulting from the disease, increasing the qualityof life of those suffering from the disease, decreasing the dose ofother medications required to treat the disease, delaying theprogression of the disease, and/or prolonging survival of patients.

As used herein, “delaying” development of Alzheimer's disease means todefer, hinder, slow, retard, stabilize, and/or postpone development ofthe disease. This delay can be of varying lengths of time, depending onthe history of the disease and/or individual being treated. As isevident to one skilled in the art, a sufficient or significant delaycan, in effect, encompass prevention, in that the individual does notdevelop the disease. A method that “delays” development of Alzheimer'sdisease is a method that reduces probability of disease development in agiven time frame and/or reduces extent of the disease in a given timeframe, when compared to not using the method. Such comparisons aretypically based on clinical studies, using a statistically significantnumber of subjects.

“Development” of Alzheimer's disease means the onset and/or progressionof Alzheimer's disease within an individual. Alzheimer's diseasedevelopment can be detectable using standard clinical techniques asdescribed herein. However, development also refers to diseaseprogression that may be initially undetectable. For purposes of thisinvention, progression refers to the biological course of the diseasestate, in this case, as determined by a standard neurologicalexamination, patient interview, or may be determined by more specializedtesting. A variety of these diagnostic tests include, but not limitedto, neuroimaging, detecting alterations of levels of specific proteinsin the serum or cerebrospinal fluid (e.g., amyloid peptides and Tau),computerized tomography (CT), and magnetic resonance imaging (MRI).“Development” includes occurrence, recurrence, and onset. As used herein“onset” or “occurrence” of Alzheimer's disease includes initial onsetand/or recurrence.

As used herein, administration “in conjunction” includes simultaneousadministration and/or administration at different times. Administrationin conjunction also encompasses administration as a co-formulation oradministration as separate compositions. As used herein, administrationin conjunction is meant to encompass any circumstance wherein an anti-Aβantibody and another agent are administered to an individual, which canoccur simultaneously and/or separately. As further discussed herein, itis understood that an anti-Aβ antibody and the other agent can beadministered at different dosing frequencies or intervals. For example,an anti-Aβ antibody can be administered weekly, while the other agentcan be administered less frequently. It is understood that the anti-Aβantibody and the other agent can be administered using the same route ofadministration or different routes of administration.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom, and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides, or embedding in a semi-solid or solid matrix forsectioning purposes. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

An “individual” (alternatively referred to as a “subject”) is a mammal,more preferably a human. Mammals also include, but are not limited to,farm animals (such as cows), sport animals, pets (such as cats, dogs,horses), primates, mice and rats.

As used herein, “vector” means a construct, which is capable ofdelivering, and preferably expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

As used herein, “pharmaceutically acceptable carrier” includes anymaterial which, when combined with an active ingredient, allows theingredient to retain biological activity and is non-reactive with thesubject's immune system. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Preferred diluents for aerosol orparenteral administration are phosphate buffered saline or normal (0.9%)saline. Compositions comprising such carriers are formulated by wellknown conventional methods (see, for example, Remington's PharmaceuticalSciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton,Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed.Mack Publishing, 2000).

The term “k_(on)”, as used herein, is intended to refer to the on rateconstant for association of an antibody to an antigen.

The term “k_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of an antibody-antigen interaction.

Compositions and Methods of Making the Compositions Antibody 9TL and 9TLDerived Antibodies and Polypeptides

This invention encompasses compositions, including pharmaceuticalcompositions, comprising antibody 9TL and its variants shown in Table 3or polypeptide derived from antibody 9TL and its variants shown in Table3; and polynucleotides comprising sequences encoding 9TL antibody andits variants or the polypeptide. As used herein, compositions compriseone or more antibodies or polypeptides (which may or may not be anantibody) that bind to C-terminus of Aβ₁₋₄₀, and/or one or morepolynucleotides comprising sequences encoding one or more antibodies orpolypeptides that bind to C-terminus of Aβ₁₋₄₀. These compositions mayfurther comprise suitable excipients, such as pharmaceuticallyacceptable excipients including buffers, which are well known in theart.

The antibodies and polypeptides of the invention are characterized byany (one or more) of the following characteristics: (a) binds toC-terminal peptide 28-40 of Aβ₁₋₄₀, but does not significantly bind toAβ1-42 or Aβ1-43; (b) binds to C-terminal peptide 33-40 of Aβ₁₋₄₀; (c)suppresses formation of amyloid plaques in a subject; (d) reducesamyloid plaques in a subject; (e) treats, prevents, ameliorates one ormore symptoms of Alzheimer's disease; (f) improves cognitive function.The antibodies and polypeptides of the invention may also exhibit adesirable safety profile in contrast to other reported anti-Aβantibodies. For example, the compositions of the invention may not causesignificant or unacceptable levels of any one or more of: bleeding inthe brain vasculature (cerebral hemorrhage); meningoencephalitis(including changing magnetic resonance scan); elevated white blood countin cerebral spinal fluid; central nervous system inflammation.

Accordingly, the invention provides any of the following, orcompositions (including pharmaceutical compositions) comprising any ofthe following: (a) antibody 9TL or its variants shown in Table 3; (b) afragment or a region of antibody 9TL or its variants shown in Table 3;(c) a light chain of antibody 9TL or its variants shown in Table 3; (d)a heavy chain of antibody 9TL or its variants shown in Table 3; (e) oneor more variable region(s) from a light chain and/or a heavy chain ofantibody 9TL or its variants shown in Table 3; (f) one or more CDR(s)(one, two, three, four, five or six CDRs) of antibody 9TL or itsvariants shown in Table 3; (g) CDR H3 from the heavy chain of antibody9TL; (h) CDR L3 from the light chain of antibody 9TL or its variantsshown in Table 3; (i) three CDRs from the light chain of antibody 9TL orits variants shown in Table 3; (j) three CDRs from the heavy chain ofantibody 9TL or its variants shown in Table 3; (k) three CDRs from thelight chain and three CDRs from the heavy chain, of antibody 9TL or itsvariants shown in Table 3; and (1) an antibody comprising any one of (b)through (k). The invention also provides polypeptides comprising any oneor more of the above.

The CDR portions of antibody 9TL (including Chothia and Kabat CDRs) arediagrammatically depicted in FIG. 1. Determination of CDR regions iswell within the skill of the art. It is understood that in someembodiments, CDRs can be a combination of the Kabat and Chothia CDR(also termed “combined CDRs” or “extended CDRs”). In some embodiments,the CDRs are the Kabat CDRs. In other embodiments, the CDRs are theChothia CDRs. In other words, in embodiments with more than one CDR, theCDRs may be any of Kabat, Chothia, combination CDRs, or combinationsthereof.

In some embodiments, the invention provides a polypeptide (which may ormay not be an antibody) which comprises at least one CDR, at least two,at least three, or at least four, at least five, or all six CDRs thatare substantially identical to at least one CDR, at least two, at leastthree, at least four, at least five or all six CDRs of 9TL or itsvariants shown in Table 3. Other embodiments include antibodies whichhave at least two, three, four, five, or six CDR(s) that aresubstantially identical to at least two, three, four, five or six CDRsof 9TL or derived from 9TL. In some embodiments, the at least one, two,three, four, five, or six CDR(s) are at least about 85%, 86%, 87%, 88%,89%, 90%, 95%, 96%, 97%, 98%, or 99% identical to at least one, two,three, four, five or six CDRs of 9TL or its variants shown in Table 3.It is understood that, for purposes of this invention, bindingspecificity and/or overall activity is generally retained, although theextent of activity may vary compared to 9TL or its variants shown inTable 3 (may be greater or lesser).

The invention also provides a polypeptide (which may or may not be anantibody) which comprises an amino acid sequence of 9TL or its variantsshown in Table 3 that has any of the following: at least 5 contiguousamino acids, at least 8 contiguous amino acids, at least about 10contiguous amino acids, at least about 15 contiguous amino acids, atleast about 20 contiguous amino acids, at least about 25 contiguousamino acids, at least about 30 contiguous amino acids of a sequence of9TL or its variants shown in Table 3, wherein at least 3 of the aminoacids are from a variable region of 9TL (FIG. 1) or its variants shownin Table 3. In one embodiment, the variable region is from a light chainof 9TL. In another embodiment, the variable region is from a heavy chainof 9TL. An exemplary polypeptide has contiguous amino acid (lengthsdescribed above) from both the heavy and light chain variable regions of9TL. In another embodiment, the 5 (or more) contiguous amino acids arefrom a complementarity determining region (CDR) of 9TL shown in FIG. 1.In some embodiments, the contiguous amino acids are from a variableregion of 9TL.

The binding affinities of the antibodies and polypeptides of theinvention may vary, and need not be (but can be) a particular value orrange, as the exemplary embodiments described below. The bindingaffinity of the antibodies and polypeptides of the invention to Aβ₁₋₄₀can be about 0.10 to about 0.80 nM, about 0.15 to about 0.75 nM andabout 0.18 to about 0.72 nM. In some embodiments, the binding affinityis about 2 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, about40 pM, or greater than about 40 pM. In one embodiment, the bindingaffinity is between about 2 pM and 22 pM. In other embodiments, thebinding affinity is less than about 10 nM, about 5 nM, about 1 nM, about900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about400 pM, about 300 pM, about 200 pM, about 150 pM, about 100 pM, about 90pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM,about 30 pM, about 10 pM. In some embodiment, the binding affinity isabout 10 nM. In other embodiments, the binding affinity is less thanabout 10 nM, less than about 50 nM, less than about 100 nM, less thanabout 150 nM, less than about 200 nM, less than about 250 nM, less thanabout 500 nM, or less than about 1000 nM. In other embodiments, thebinding affinity is less than about 5 nM. In other embodiments, thebinding affinity is less than about 1 nM. In other embodiments, thebinding affinity is about 0.1 nM or about 0.07 nM. In other embodiments,the binding affinity is less than about 0.1 nM or less than about 0.07nM. In other embodiments, the binding affinity is from any of about 10nM, about 5 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM,about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM,about 150 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about60 pM, about 50 pM, about 40 pM, about 30 pM, about 10 pM to any ofabout 2 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, or about40 pM. In some embodiments, the binding affinity is any of about 10 nM,about 5 nM, about 1 nM, about 900 pM, about 800 pM, bout 700 pM, about600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about150 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60pM, about 50 pM, about 40 pM, about 30 pM, about 10 pM. In still otherembodiments, the binding affinity is about 2 pM, about 5 pM, about 10pM, about 15 pM, about 20 pM, about 40 pM, or greater than about 40 pM.

The invention also provides methods of making any of these antibodies orpolypeptides. The antibodies of this invention can be made by proceduresknown in the art. The polypeptides can be produced by proteolytic orother degradation of the antibodies, by recombinant methods (i.e.,single or fusion polypeptides) as described above or by chemicalsynthesis. Polypeptides of the antibodies, especially shorterpolypeptides up to about 50 amino acids, are conveniently made bychemical synthesis. Methods of chemical synthesis are known in the artand are commercially available. For example, an antibody could beproduced by an automated polypeptide synthesizer employing the solidphase method. See also, U.S. Pat. Nos. 5,807,715; 4,816,567; and6,331,415.

In another alternative, the antibodies can be made recombinantly usingprocedures that are well known in the art. In one embodiment, apolynucleotide comprises a sequence encoding the heavy chain and/or thelight chain variable regions of antibody 9TL shown in SEQ ID NO:9 andSEQ ID NO:10. In another embodiment, the polynucleotide comprising thenucleotide sequence shown in SEQ ID NO:9 and SEQ ID NO:10 are clonedinto one or more vectors for expression or propagation. The sequenceencoding the antibody of interest may be maintained in a vector in ahost cell and the host cell can then be expanded and frozen for futureuse. Vectors (including expression vectors) and host cells are furtherdescribed herein.

The invention also encompasses single chain variable region fragments(“scFv”) of antibodies of this invention, such as 9TL. Single chainvariable region fragments are made by linking light and/or heavy chainvariable regions by using a short linking peptide. Bird et al. (1988)Science 242:423-426. An example of a linking peptide is (GGGGS)₃ (SEQ IDNO:40) which bridges approximately 3.5 nm between the carboxy terminusof one variable region and the amino terminus of the other variableregion. Linkers of other sequences have been designed and used. Bird etal. (1988). Linkers can in turn be modified for additional functions,such as attachment of drugs or attachment to solid supports. The singlechain variants can be produced either recombinantly or synthetically.For synthetic production of scFv, an automated synthesizer can be used.For recombinant production of scFv, a suitable plasmid containingpolynucleotide that encodes the scFv can be introduced into a suitablehost cell, either eukaryotic, such as yeast, plant, insect or mammaliancells, or prokaryotic, such as E. coli. Polynucleotides encoding thescFv of interest can be made by routine manipulations such as ligationof polynucleotides. The resultant scFv can be isolated using standardprotein purification techniques known in the art.

Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

For example, bispecific antibodies, monoclonal antibodies that havebinding specificities for at least two different antigens, can beprepared using the antibodies disclosed herein. Methods for makingbispecific antibodies are known in the art (see, e.g., Suresh et al.,1986, Methods in Enzymology 121:210). Traditionally, the recombinantproduction of bispecific antibodies was based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, with the two heavy chainshaving different specificities (Millstein and Cuello, 1983, Nature 305,537-539).

According to one approach to making bispecific antibodies, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulin heavychain constant domain, comprising at least part of the hinge, CH2 andCH3 regions. It is preferred to have the first heavy chain constantregion (CH1), containing the site necessary for light chain binding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are cotransfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In one approach, the bispecific antibodies are composed of a hybridimmunoglobulin heavy chain with a first binding specificity in one arm,and a hybrid immunoglobulin heavy chain-light chain pair (providing asecond binding specificity) in the other arm. This asymmetric structure,with an immunoglobulin light chain in only one half of the bispecificmolecule, facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations. This approach isdescribed in PCT Publication No. WO 94/04690, published Mar. 3, 1994.

Heteroconjugate antibodies, comprising two covalently joined antibodies,are also within the scope of the invention. Such antibodies have beenused to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (PCT applicationpublication Nos. WO 91/00360 and WO 92/200373; EP 03089).Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents and techniques arewell known in the art, and are described in U.S. Pat. No. 4,676,980.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods of synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Humanized antibody comprising one or more CDRs of antibody 9TL or one ormore CDRs derived from antibody 9TL can be made using any methods knownin the art. For example, four general steps may be used to humanize amonoclonal antibody. These are: (1) determining the nucleotide andpredicted amino acid sequence of the starting antibody light and heavyvariable domains (2) designing the humanized antibody, i.e., decidingwhich antibody framework region to use during the humanizing process (3)the actual humanizing methodologies/techniques and (4) the transfectionand expression of the humanized antibody. See, for example, U.S. Pat.Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761;5,693,762; 5,585,089; 6,180,370; 5,225,539; 6,548,640.

In the recombinant humanized antibodies, the Fc portion can be modifiedto avoid interaction with Fcγ receptor and the complement immune system.This type of modification was designed by Dr. Mike Clark from theDepartment of Pathology at Cambridge University, and techniques forpreparation of such antibodies are described in WO 99/58572, publishedNov. 18, 1999.

For example, the constant region may be engineered to more resemblehuman constant regions to avoid immune response if the antibody for usein clinical trials and treatments in humans. See, for example, U.S. Pat.Nos. 5,997,867 and 5,866,692.

The invention encompasses modifications to antibody 9TL, includingfunctionally equivalent antibodies which do not significantly affecttheir properties and variants which have enhanced or decreased activityand/or affinity. For example, the amino acid sequence of antibody 9TLmay be mutated to obtain an antibody with the desired binding affinityto Aβ₁₋₄₀ peptide. Modification of polypeptides is routine practice inthe art and need not be described in detail herein. Modification ofpolypeptides is exemplified in the Examples. Examples of modifiedpolypeptides include polypeptides with conservative substitutions ofamino acid residues, one or more deletions or additions of amino acidswhich do not significantly deleteriously change the functional activity,or use of chemical analogs.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto an epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the serum half-life of the antibody.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 1 under the heading of“conservative substitutions”. If such substitutions result in a changein biological activity, then more substantial changes, denominated“exemplary substitutions” in Table 1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 1 Amino Acid Substitutions Conservative Original ResidueSubstitutions Exemplary Substitutions Ala (A) Val Val; Leu; Ile Arg (R)Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu;Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp; Gln Gly(G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met;Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val;Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W)Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met;Phe; Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile;

(2) Polar without charge: Cys, Ser, Thr, Asn, Gln;

(3) Acidic (negatively charged): Asp, Glu;

(4) Basic (positively charged): Lys, Arg;

(5) Residues that influence chain orientation: Gly, Pro; and

(6) Aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions are made by exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcross-linking Conversely, cysteine bond(s) may be added to the antibodyto improve its stability, particularly where the antibody is an antibodyfragment such as an Fv fragment.

Amino acid modifications can range from changing or modifying one ormore amino acids to complete redesign of a region, such as the variableregion. Changes in the variable region can alter binding affinity and/orspecificity. In some embodiments, no more than one to five conservativeamino acid substitutions are made within a CDR domain. In otherembodiments, no more than one to three conservative amino acidsubstitutions are made within a CDR domain. In still other embodiments,the CDR domain is CDR H3 and/or CDR L3.

Modifications also include glycosylated and nonglycosylatedpolypeptides, as well as polypeptides with other post-translationalmodifications, such as, for example, glycosylation with differentsugars, acetylation, and phosphorylation. Antibodies are glycosylated atconserved positions in their constant regions (Jefferis and Lund, 1997,Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32).The oligosaccharide side chains of the immunoglobulins affect theprotein's function (Boyd et al., 1996, Mol. Immunol. 32:1311-1318;Wittwe and Howard, 1990, Biochem. 29:4175-4180) and the intramolecularinteraction between portions of the glycoprotein, which can affect theconformation and presented three-dimensional surface of the glycoprotein(Hefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.7:409-416). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular, CHOcells with tetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al., 1999, MatureBiotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

The glycosylation pattern of antibodies may also be altered withoutaltering the underlying nucleotide sequence. Glycosylation largelydepends on the host cell used to express the antibody. Since the celltype used for expression of recombinant glycoproteins, e.g. antibodies,as potential therapeutics is rarely the native cell, variations in theglycosylation pattern of the antibodies can be expected (see, e.g. Hseet al., 1997, J. Biol. Chem. 272:9062-9070).

In addition to the choice of host cells, factors that affectglycosylation during recombinant production of antibodies include growthmode, media formulation, culture density, oxygenation, pH, purificationschemes and the like. Various methods have been proposed to alter theglycosylation pattern achieved in a particular host organism includingintroducing or overexpressing certain enzymes involved inoligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and5,278,299). Glycosylation, or certain types of glycosylation, can beenzymatically removed from the glycoprotein, for example usingendoglycosidase H (Endo H), N-glycosidase F as described in Example 3,endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. In addition,the recombinant host cell can be genetically engineered to be defectivein processing certain types of polysaccharides. These and similartechniques are well known in the art.

Other methods of modification include using coupling techniques known inthe art, including, but not limited to, enzymatic means, oxidativesubstitution and chelation. Modifications can be used, for example, forattachment of labels for immunoassay. Modified 9TL polypeptides are madeusing established procedures in the art and can be screened usingstandard assays known in the art, some of which are described below andin the Examples.

In some embodiments of the invention, the antibody comprises a modifiedconstant region, such as a constant region that is immunologically inertor partially inert, e.g., does not trigger complement mediated lysis,does not stimulate antibody-dependent cell mediated cytotoxicity (ADCC),or does not activate microglia; or have reduced activities (compared tothe unmodified antibody) in any one or more of the following: triggeringcomplement mediated lysis, stimulating antibody-dependent cell mediatedcytotoxicity (ADCC), or activating microglia. Different modifications ofthe constant region may be used to achieve optimal level and/orcombination of effector functions. See, for example, Morgan et al.,Immunology 86:319-324 (1995); Lund et al., J. Immunology 157:4963-9157:4963-4969 (1996); Idusogie et al., J. Immunology 164:4178-4184(2000); Tao et al., J. Immunology 143: 2595-2601 (1989); and Jefferis etal., Immunological Reviews 163:59-76 (1998). In some embodiments, theconstant region is modified as described in Eur. J. Immunol. (1999)29:2613-2624; PCT Application No. PCT/GB99/01441; and/or UK PatentApplication No. 9809951.8. In other embodiments, the antibody comprisesa human heavy chain IgG2a constant region comprising the followingmutations: A330P331 to S330S331 (amino acid numbering with reference tothe wildtype IgG2a sequence). Eur. J. Immunol. (1999) 29:2613-2624. Instill other embodiments, the constant region is aglycosylated forN-linked glycosylation. In some embodiments, the constant region isaglycosylated for N-linked glycosylation by mutating the glycosylatedamino acid residue or flanking residues that are part of theN-glycosylation recognition sequence in the constant region. Forexample, N-glycosylation site N297 may be mutated to A, Q, K, or H. See,Tao et al., J. Immunology 143: 2595-2601 (1989); and Jefferis et al.,Immunological Reviews 163:59-76 (1998). In some embodiments, theconstant region is aglycosylated for N-linked glycosylation. Theconstant region may be aglycosylated for N-linked glycosylationenzymatically (such as removing carbohydrate by enzyme PNGase), or byexpression in a glycosylation deficient host cell.

Other antibody modifications include antibodies that have been modifiedas described in PCT Publication No. WO 99/58572, published Nov. 18,1999. These antibodies comprise, in addition to a binding domaindirected at the target molecule, an effector domain having an amino acidsequence substantially homologous to all or part of a constant domain ofa human immunoglobulin heavy chain. These antibodies are capable ofbinding the target molecule without triggering significant complementdependent lysis, or cell-mediated destruction of the target. In someembodiments, the effector domain is capable of specifically binding FcRnand/or FcγRIIb. These are typically based on chimeric domains derivedfrom two or more human immunoglobulin heavy chain C_(H)2 domains.Antibodies modified in this manner are particularly suitable for use inchronic antibody therapy, to avoid inflammatory and other adversereactions to conventional antibody therapy.

The invention includes affinity matured embodiments. For example,affinity matured antibodies can be produced by procedures known in theart (Marks et al., 1992, Bio/Technology, 10:779-783; Barbas et al.,1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier et al., 1995, Gene,169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; Jackson etal., 1995, J. Immunol., 154(7):3310-9; Hawkins et al, 1992, J. Mol.Biol., 226:889-896; and WO2004/058184).

The following methods may be used for adjusting the affinity of anantibody and for characterizing a CDR. One way of characterizing a CDRof an antibody and/or altering (such as improving) the binding affinityof a polypeptide, such as an antibody, termed “library scanningmutagenesis”. Generally, library scanning mutagenesis works as follows.One or more amino acid positions in the CDR are replaced with two ormore (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20) amino acids using art recognized methods. This generatessmall libraries of clones (in some embodiments, one for every amino acidposition that is analyzed), each with a complexity of two or moremembers (if two or more amino acids are substituted at every position).Generally, the library also includes a clone comprising the native(unsubstituted) amino acid. A small number of clones, e.g., about 20-80clones (depending on the complexity of the library), from each libraryare screened for binding affinity to the target polypeptide (or otherbinding target), and candidates with increased, the same, decreased orno binding are identified. Methods for determining binding affinity arewell-known in the art. Binding affinity may be determined using BIAcoresurface plasmon resonance analysis, which detects differences in bindingaffinity of about 2-fold or greater. BIAcore is particularly useful whenthe starting antibody already binds with a relatively high affinity, forexample a K_(D) of about 10 nM or lower. Screening using BIAcore surfaceplasmon resonance is described in the Examples, herein.

Binding affinity may be determined using Kinexa Biocensor, scintillationproximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescencequenching, fluorescence transfer, and/or yeast display. Binding affinitymay also be screened using a suitable bioassay.

In some embodiments, every amino acid position in a CDR is replaced (insome embodiments, one at a time) with all 20 natural amino acids usingart recognized mutagenesis methods (some of which are described herein).This generates small libraries of clones (in some embodiments, one forevery amino acid position that is analyzed), each with a complexity of20 members (if all 20 amino acids are substituted at every position).

In some embodiments, the library to be screened comprises substitutionsin two or more positions, which may be in the same CDR or in two or moreCDRs. Thus, the library may comprise substitutions in two or morepositions in one CDR. The library may comprise substitution in two ormore positions in two or more CDRs. The library may comprisesubstitution in 3, 4, 5, or more positions, said positions found in two,three, four, five or six CDRs. The substitution may be prepared usinglow redundancy codons. See, e.g., Table 2 of Balint et al., (1993) Gene137(1):109-18).

The CDR may be CDRH3 and/or CDRL3. The CDR may be one or more of CDRL1,CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH3. The CDR may be a Kabat CDR, aChothia CDR, or an extended CDR.

Candidates with improved binding may be sequenced, thereby identifying aCDR substitution mutant which results in improved affinity (also termedan “improved” substitution). Candidates that bind may also be sequenced,thereby identifying a CDR substitution which retains binding.

Multiple rounds of screening may be conducted. For example, candidates(each comprising an amino acid substitution at one or more position ofone or more CDR) with improved binding are also useful for the design ofa second library containing at least the original and substituted aminoacid at each improved CDR position (i.e., amino acid position in the CDRat which a substitution mutant showed improved binding). Preparation,and screening or selection of this library is discussed further below.

Library scanning mutagenesis also provides a means for characterizing aCDR, in so far as the frequency of clones with improved binding, thesame binding, decreased binding or no binding also provide informationrelating to the importance of each amino acid position for the stabilityof the antibody-antigen complex. For example, if a position of the CDRretains binding when changed to all 20 amino acids, that position isidentified as a position that is unlikely to be required for antigenbinding. Conversely, if a position of CDR retains binding in only asmall percentage of substitutions, that position is identified as aposition that is important to CDR function. Thus, the library scanningmutagenesis methods generate information regarding positions in the CDRsthat can be changed to many different amino acid (including all 20 aminoacids), and positions in the CDRs which cannot be changed or which canonly be changed to a few amino acids.

Candidates with improved affinity may be combined in a second library,which includes the improved amino acid, the original amino acid at thatposition, and may further include additional substitutions at thatposition, depending on the complexity of the library that is desired, orpermitted using the desired screening or selection method. In addition,if desired, adjacent amino acid position can be randomized to at leasttwo or more amino acids. Randomization of adjacent amino acids maypermit additional conformational flexibility in the mutant CDR, whichmay in turn, permit or facilitate the introduction of a larger number ofimproving mutations. The library may also comprise substitution atpositions that did not show improved affinity in the first round ofscreening.

The second library is screened or selected for library members withimproved and/or altered binding affinity using any method known in theart, including screening using BIAcore surface plasmon resonanceanalysis, and selection using any method known in the art for selection,including phage display, yeast display, and ribosome display.

The invention also encompasses fusion proteins comprising one or morefragments or regions from the antibodies (such as 9TL) or polypeptidesof this invention. In one embodiment, a fusion polypeptide is providedthat comprises at least 10 contiguous amino acids of the variable lightchain region shown in SEQ ID NO:2 (FIG. 1) and/or at least 10 aminoacids of the variable heavy chain region shown in SEQ ID NO:1 (FIG. 1).In other embodiments, a fusion polypeptide is provided that comprises atleast about 10, at least about 15, at least about 20, at least about 25,or at least about 30 contiguous amino acids of the variable light chainregion shown in SEQ ID NO:2 (FIG. 1) and/or at least about 10, at leastabout 15, at least about 20, at least about 25, or at least about 30contiguous amino acids of the variable heavy chain region shown in SEQID NO:1 (FIG. 1). In another embodiment, the fusion polypeptidecomprises a light chain variable region and/or a heavy chain variableregion of 9TL, as shown in SEQ ID NO:2 and SEQ ID NO:1 of FIG. 1. Inanother embodiment, the fusion polypeptide comprises one or more CDR(s)of 9TL. In still other embodiments, the fusion polypeptide comprises CDRH3 and/or CDR L3 of antibody 9TL. For purposes of this invention, an 9TLfusion protein contains one or more 9TL antibodies and another aminoacid sequence to which it is not attached in the native molecule, forexample, a heterologous sequence or a homologous sequence from anotherregion. Exemplary heterologous sequences include, but are not limited toa “tag” such as a FLAG tag or a 6H is tag. Tags are well known in theart.

A 9TL fusion polypeptide can be created by methods known in the art, forexample, synthetically or recombinantly. Typically, the 9TL fusionproteins of this invention are made by preparing an expressing apolynucleotide encoding them using recombinant methods described herein,although they may also be prepared by other means known in the art,including, for example, chemical synthesis.

This invention also provides compositions comprising 9TL antibodies orpolypeptides conjugated (for example, linked) to an agent thatfacilitate coupling to a solid support (such as biotin or avidin). Forsimplicity, reference will be made generally to 9TL or antibodies withthe understanding that these methods apply to any of the Aβ₁₋₄₀ bindingembodiments described herein. Conjugation generally refers to linkingthese components as described herein. The linking (which is generallyfixing these components in proximate association at least foradministration) can be achieved in any number of ways. For example, adirect reaction between an agent and an antibody is possible when eachpossesses a substituent capable of reacting with the other. For example,a nucleophilic group, such as an amino or sulfhydryl group, on one maybe capable of reacting with a carbonyl-containing group, such as ananhydride or an acid halide, or with an alkyl group containing a goodleaving group (e.g., a halide) on the other.

An antibody or polypeptide of this invention may be linked to a labelingagent (alternatively termed “label”) such as a fluorescent molecule, aradioactive molecule or any others labels known in the art. Labels areknown in the art which generally provide (either directly or indirectly)a signal.

The invention also provides compositions (including pharmaceuticalcompositions) and kits comprising antibody 9TL, and, as this disclosuremakes clear, any or all of the antibodies and/or polypeptides describedherein.

Anti-Aβ Peptide Antibodies and Polypeptides Having Impaired EffectorFunction

The methods of the invention use antibodies or polypeptides (includingpharmaceutical compositions comprising the antibodies or polypeptides)that specifically bind to a beta-amyloid peptide and have impairedeffector function. The antibodies and polypeptides are furthercharacterized by any (one or more) of the following characteristics: (a)suppresses formation of amyloid plaques in a subject; (b) reducesamyloid plaques in a subject; (c) treats, prevents, ameliorates one ormore symptoms of Alzheimer's disease; (d) improves cognitive function.The antibodies and polypeptides described herein may exhibit a desirablesafety profile, for example, the compositions of the invention do notcause significant or unacceptable levels or have a reduced level of anyone or more of: bleeding in the brain vasculature (cerebral hemorrhage);meningoencephalitis (including changing magnetic resonance scan);elevated white blood count in cerebral spinal fluid; central nervoussystem inflammation. As shown in Example 4, an anti-Aβ antibody havingN-linked glycosylation removed in the Fc region was effective inremoving amyloid plaques in the brain and improving cognitive functionwith significantly less microhemorrhage than the intact antibody in ananimal model for Alzheimer's disease.

As used herein, an antibody or a polypeptide having an “impairedeffector function” (used interchangeably with “immunologically inert” or“partially immunologically inert”) refers to antibodies or polypeptidesthat do not have any effector function or have reduced activity oractivities of effector function (compared to antibody or polypeptidehaving an unmodified or a naturally occurring constant region), e.g.,having no activity or reduced activity in any one or more of thefollowing: a) triggering complement mediated lysis; b) stimulatingantibody-dependent cell mediated cytotoxicity (ADCC); and c) activatingmicroglia. The effector function activity may be reduced by about any of10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, and 100%. In someembodiments, the antibody binds to a beta-amyloid peptide withouttriggering significant complement dependent lysis, or cell mediateddestruction of the target. For example, the Fc receptor binding site onthe constant region may be modified or mutated to remove or reducebinding affinity to certain Fc receptors, such as FcγRI, FcγRII, and/orFcγRIII. For simplicity, reference will be made to antibodies with theunderstanding that embodiments also apply to polypeptides. EU numberingsystem (Kabat et al., Sequences of Proteins of Immunological Interest;5th ed. Public Health Service, National Institutes of Healthy, Bethesda,Md., 1991) is used to indicate which amino acid residue(s) of theconstant region (e.g., of an IgG antibody) are altered or mutated. Thenumbering may be used for a specific type of antibody (e.g., IgG1) or aspecies (e.g., human) with the understanding that similar changes can bemade across types of antibodies and species.

In some embodiments, the antibody that specifically binds to the an Aβpeptide comprises a heavy chain constant region having impaired effectorfunction. The heavy chain constant region may have naturally occurringsequence or is a variant. In some embodiments, the amino acid sequenceof a naturally occurring heavy chain constant region is mutated, e.g.,by amino acid substitution, insertion and/or deletion, whereby theeffector function of the constant region is impaired. In someembodiments, the N-glycosylation of the Fc region of a heavy chainconstant region may also be changed, e.g., may be removed completely orpartially, whereby the effector function of the constant region isimpaired.

In some embodiments, the effector function is impaired by removingN-glycosylation of the Fc region (e.g., in the CH 2 domain of IgG) ofthe anti-Aβ peptide. In some embodiments, N-glycosylation of the Fcregion is removed by mutating the glycosylated amino acid residue orflanking residues that are part of the glycosylation recognitionsequence in the constant region. The tripeptide sequencesasparagine-X-serine (N-X-S), asparagine-X-threonine (N-X-T) andasparagine-X-cysteine (N-X-C), where X is any amino acid except proline,are the recognition sequences for enzymatic attachment of thecarbohydrate moiety to the asparagine side chain for N-glycosylation.Mutating any of the amino acid in the tripeptide sequences in theconstant region yields an aglycosylated IgG. For example,N-glycosylation site N297 of human IgG1 and IgG3 may be mutated to A, D,Q, K, or H. See, Tao et al., J. Immunology 143: 2595-2601 (1989); andJefferis et al., Immunological Reviews 163:59-76 (1998). It has beenreported that human IgG1 and IgG3 with substitution of Asn-297 with Gln,H is, or Lys do not bind to the human FcγRI and do not activatecomplement with Clq binding ability completely lost for IgG1 anddramatically decreased for IgG3. In some embodiments, the amino acid Nin the tripeptide sequences is mutated to any one of amino acid A, C, D,E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y. In some embodiments, theamino acid N in the tripeptide sequences is mutated to a conservativesubstitution. In some embodiments, the amino acid X in the tripeptidesequences is mutated to proline. In some embodiments, the amino acid Sin the tripeptide sequences is mutated to A, D, E, F, G, H, I, K, L, M,N, P, Q, R, V, W, Y. In some embodiments, the amino acid T in thetripeptide sequences is mutated to A, D, E, F, G, H, I, K, L, M, N, P,Q, R, V, W, Y. In some embodiments, the amino acid C in the tripeptidesequences is mutated to A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W,Y. In some embodiments, the amino acid following the tripeptide ismutated to P. In some embodiments, the N-glycosylation in the constantregion is removed enzymatically (such as N-glycosidase F as described inExample 3, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3,and englycosidase H). Removing N-glycosylation may also be achieved byproducing the antibody in a cell line having deficiency forN-glycosylation. Wright et al., J. Immunol. 160(7):3393-402 (1998).

In some embodiments, amino acid residue interacting with oligosaccharideattached to the N-glycosylation site of the constant region is mutatedto reduce binding affinity to FcγRI. For example, F241, V264, D265 ofhuman IgG3 may be mutated. See, Lund et al., J. Immunology 157:4963-4969(1996).

In some embodiments, the effector function is impaired by modifyingregions such as 233-236, 297, and/or 327-331 of human IgG as describedin PCT WO 99/58572 and Armour et al., Molecular Immunology 40: 585-593(2003); Reddy et al., J. Immunology 164:1925-1933 (2000). Antibodiesdescribed in PCT WO 99/58572 and Armour et al. comprise, in addition toa binding domain directed at the target molecule, an effector domainhaving an amino acid sequence substantially homologous to all or part ofa constant region of a human immunoglobulin heavy chain. Theseantibodies are capable of binding the target molecule without triggeringsignificant complement dependent lysis, or cell-mediated destruction ofthe target. In some embodiments, the effector domain has a reducedaffinity for FcγRI, FcγRIIa, and FcγRIII. In some embodiments, theeffector domain is capable of specifically binding FcRn and/or FcγRIIb.These are typically based on chimeric domains derived from two or morehuman immunoglobulin heavy chain C_(H)2 domains. Antibodies modified inthis manner are particularly suitable for use in chronic antibodytherapy, to avoid inflammatory and other adverse reactions toconventional antibody therapy. In some embodiments, the heavy chainconstant region of the antibody is a human heavy chain IgG1 with any ofthe following mutations: 1) A327A330P331 to G327S330S331; 2)E233L234L235G236 to P233V234A235 with G236 deleted; 3) E233L234L235 toP233V234A235; 4) E233L234L235G236A327A330P331 toP233V234A235G327S330S331 with G236 deleted; 5) E233L234L235A327A330P331to P233V234A235G327S330S331; and 6) N297 to A297 or any other amino acidexcept N. In some embodiments, the heavy chain constant region of theantibody is a human heavy chain IgG2 with the following mutations:A330P331 to S330S331. In some embodiments, the heavy chain constantregion of the antibody is a human heavy chain IgG4 with any of thefollowing mutations: E233F234L235G236 to P233V234A235 with G236 deleted;E233F234L235 to P233V234A235; and S228L235 to P228E235.

The constant region of the antibodies may also be modified to impaircomplement activation. For example, complement activation of IgGantibodies following binding of the Cl component of complement may bereduced by mutating amino acid residues in the constant region in a Clbinding motif (e.g., Clq binding motif). It has been reported that Alamutation for each of D270, K322, P329, P331 of human IgG1 significantlyreduced the ability of the antibody to bind to Clq and activatingcomplement. For murine IgG2b, Clq binding motif constitutes residuesE318, K320, and K322. Idusogie et al., J. Immunology 164:4178-4184(2000); Duncan et al., Nature 322: 738-740 (1988).

Clq binding motif E318, K320, and K322 identified for murine IgG2b isbelieved to be common for other antibody isotypes. Duncan et al., Nature322: 738-740 (1988). Clq binding activity for IgG2b can be abolished byreplacing any one of the three specified residues with a residue havingan inappropriate functionality on its side chain. It is not necessary toreplace the ionic residues only with Ala to abolish Clq binding. It isalso possible to use other alkyl-substituted non-ionic residues, such asGly, Ile, Leu, or Val, or such aromatic non-polar residues as Phe, Tyr,Trp and Pro in place of any one of the three residues in order toabolish Clq binding. In addition, it is also be possible to use suchpolar non-ionic residues as Ser, Thr, Cys, and Met in place of residues320 and 322, but not 318, in order to abolish Clq binding activity.

The invention also provides antibodies having impaired effector functionwherein the antibody has a modified hinge region. Binding affinity ofhuman IgG for its Fc receptors can be modulated by modifying the hingeregion. Canfield et al., J. Exp. Med. 173:1483-1491 (1991); Hezareh etal., J. Virol. 75:12161-12168 (2001); Redpath et al., Human Immunology59:720-727 (1998). Specific amino acid residues may be mutated ordeleted. The modified hinge region may comprise a complete hinge regionderived from an antibody of different antibody class or subclass fromthat of the CH1 domain. For example, the constant domain (CH1) of aclass IgG antibody can be attached to a hinge region of a class IgG4antibody. Alternatively, the new hinge region may comprise part of anatural hinge or a repeating unit in which each unit in the repeat isderived from a natural hinge region. In some embodiments, the naturalhinge region is altered by converting one or more cysteine residues intoa neutral residue, such as alanine, or by converting suitably placedresidues into cysteine residues. U.S. Pat. No. 5,677,425. Suchalterations are carried out using art recognized protein chemistry and,preferably, genetic engineering techniques and as described herein.

Polypeptides that specifically bind to an Aβ peptide and fused to aheavy chain constant region having impaired effector function may alsobe used for the methods described herein. In some embodiments, thepolypeptide comprises a sequence derived from antibody 9TL or itsvariants shown in Table 3. In some embodiments, the polypeptide isderived from a single domain antibody that binds to an Aβ peptide.Single domain antibodies can be generated using methods known in theart. Omidfar et al., Tumour Biol. 25:296-305 (2004); Herring et al.,Trends in Biotechnology 21:484-489 (2003).

In some embodiments, the antibody or polypeptide is not a F(ab′)₂fragment. In some embodiments, the antibody or polypeptide is not a Fabfragment. In some embodiments, the antibody or polypeptide is not asingle chain antibody scFv. In some embodiments, the antibody orpolypeptide is a PEGylated F(ab′)₂ fragment. In some embodiments, theantibody or polypeptide is a PEGylated Fab fragment. In someembodiments, the antibody or polypeptide is a PEGylated single chainantibody scFv.

Other methods to make antibodies having impaired effector function knownin the art may also be used.

Antibodies and polypeptides with modified constant regions can be testedin one or more assays to evaluate level of effector function reductionin biological activity compared to the starting antibody. For example,the ability of the antibody or polypeptide with an altered Fc region tobind complement or Fc receptors (for example, Fc receptors onmicroglia), or altered hinge region can be assessed using the assaysdisclosed herein as well as any art recognized assay. PCT WO 99/58572;Armour et al., Molecular Immunology 40: 585-593 (2003); Reddy et al., J.Immunology 164:1925-1933 (2000); Song et al., Infection and Immunity70:5177-5184 (2002).

In some embodiments, the antibody that specifically binds tobeta-amyloid peptide is a polyclonal antibody. In some embodiments, theantibody is a monoclonal antibody. In some embodiments, the antibody isa human antibody. In some embodiments, the antibody is a chimericantibody. In some embodiments, the antibody is a humanized antibody. Insome embodiments, the antibody is a primatized antibody. See, e.g.,Yocum et al., J. Rheumatol. 25:1257-62 (1998); Bugelski et al., Human &Experimental Toxicology 19:230-243 (2000). In some embodiments, theantibody is deimmunized by mutation so that the antibody does notactivate human immune system. See, e.g., Nanus, et al., J. Urology170:S84-S89 (2003).

As used herein, Aβ peptide includes any fragments of the enzymaticcleavage products of amyloid precursor protein. For example, Aβ peptideincludes any fragments of Aβ₁₋₄₀, Aβ₁₋₄₂, or Aβ₁₋₄₃; and peptides whichare truncated with various number of amino acids at the N-terminus orthe C-terminus of Aβ₁₋₄₀, Aβ₁₋₄₂, or Aβ₁₋₄₃. Amino acid numbering usedherein is based on the numbering for Aβ1-43 (SEQ ID NO:17).

In some embodiments, the antibody or polypeptide specifically binds toan epitope within residues 1-16 of Aβ peptide. In some embodiments, theantibody or polypeptide specifically binds to an epitope within residues16-28 of Aβ peptide. In some embodiments, the antibody or polypeptidespecifically binds to an epitope within residues 28-40 of Aβ₁₋₄₀peptide. In some embodiments, the antibody or polypeptide specificallybinds to an epitope within residues 28-42 of Aβ₁₋₄₂ peptide. In someembodiments, the antibody or polypeptide specifically binds to anepitope within residues 28-43 of Aβ₁₋₄₃ peptide. In some embodiments,the antibody or polypeptide specifically binds to an Aβ peptide withoutbinding to full-length amyloid precursor protein (APP). In someembodiments, the antibody or the polypeptide specifically binds to theaggregated form of Aβ without binding to the soluble form. In someembodiments, the antibody or the polypeptide specifically binds to thesoluble form of Aβ without binding to the aggregated form. In someembodiments, the antibody or the polypeptide specifically binds to bothaggregated form and soluble forms of Aβ. Antibodies that bind to variousaggregated form of Aβ are known in the art, for example, antibodies thatbind to amyloid beta-derived diffusible ligands (ADDLs); antibodies thatbind to amyloid fibrils and/or deposit. WO 03/104437; U.S. Pub. No.2003/0147887; U.S. Pub. No. 2004/0219146.

In some embodiments, the antibody or polypeptide comprises one, two, orthree CDRs from the 3D6 immunoglobulin light chain (SEQ ID NO:2 in U.S.Pub. Nos. 2003/0165496, or 2004/0087777), and/or one, two, or three CDRsfrom the 3D6 immunoglobulin heavy chain (SEQ ID NO:4 in U.S. Pub. Nos.2003/0165496, or 2004/0087777). In some embodiments, the antibody orpolypeptide comprises a variable heavy chain region as set forth in SEQID NO:8 in U.S. Pub. No. 2003/0165496 and a variable light chain regionas set forth in SEQ ID NO:5 in U.S. Pub. No. 2003/0165496. In someembodiments, the antibody or polypeptide comprises a variable heavychain region as set forth in SEQ ID NO:12 in U.S. Pub. No. 2003/0165496and a variable light chain region as set forth in SEQ ID NO:11 in U.S.Pub. No. 2003/0165496. In some embodiments, the antibody or polypeptidecomprises one, two, or three CDRs from the 10D5 immunoglobulin lightchain (SEQ ID NO:14 in U.S. Pub. Nos. 2003/0165496, or 2004/0087777),and/or one, two, or three CDRs from the 10D5 immunoglobulin heavy chain(SEQ ID NO:16 in U.S. Pub. Nos. 2003/0165496, or 2004/0087777).

In some embodiments, the antibody or polypeptide specifically binds toan epitope within residues 33-40 of Aβ₁₋₄₀. In some embodiments, theantibody or polypeptide specifically binds to an epitope on Aβ₁₋₄₀ thatincludes amino acid 35-40. In some embodiments, the antibody orpolypeptide specifically binds to an epitope on Aβ₁₋₄₀ that includesamino acid 36-40. In some embodiments, the antibody or polypeptidespecifically binds to an epitope on Aβ₁₋₄₀ that includes amino acid 39and/or 40. In some embodiments, the antibody or polypeptide specificallybinds to Aβ₁₋₄₀ but does not specifically bind to Aβ₁₋₄₂ and/or Aβ₁₋₄₃.In some embodiments, the antibody or polypeptide is antibody 9TL or anantibody or a polypeptide derived from 9TL described herein. In someembodiments, the antibody or polypeptide competitively inhibits bindingof antibody 9TL and/or antibody or polypeptide derived from 9TL toAβ₁₋₄₀. In some embodiments, the antibody is not antibody 2286 describedin PCT WO 2004/032868.

The binding affinities of the antibodies and polypeptides of theinvention may vary, and need not be (but can be) a particular value orrange, as the exemplary embodiments described below. The bindingaffinity of the antibodies and polypeptides of the invention to Aβ₁₋₄₀,Aβ₁₋₄₀, or Aβ₁₋₄₀ can be about 0.10 to about 0.80 nM, about 0.15 toabout 0.75 nM and about 0.18 to about 0.72 nM. In some embodiments, thebinding affinity is about 2 pM, about 5 pM, about 10 pM, about 15 pM,about 20 pM, about 40 pM, or greater than about 40 pM. In oneembodiment, the binding affinity is between about 2 pM and 22 pM. Inother embodiments, the binding affinity is less than about 10 nM, about5 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 150pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM,about 50 pM, about 40 pM, about 30 pM, about 10 pM. In some embodiments,the binding affinity is about 10 nM. In other embodiments, the bindingaffinity is less than about 10 nM, less than about 50 nM, less thanabout 100 nM, less than about 150 nM, less than about 200 nM, less thanabout 250 nM, less than about 500 nM, or less than about 1000 nM. Inother embodiments, the binding affinity is less than about 5 nM. Inother embodiments, the binding affinity is less than about 1 nM. Inother embodiments, the binding affinity is about 0.1 nM or about 0.07nM. In other embodiments, the binding affinity is less than about 0.1 nMor less than about 0.07 nM. In other embodiments, the binding affinityis from any of about 10 nM, about 5 nM, about 1 nM, about 900 pM, about800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about300 pM, about 200 pM, about 150 pM, about 100 pM, about 90 pM, about 80pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM,about 10 pM to any of about 2 pM, about 5 pM, about 10 pM, about 15 pM,about 20 pM, or about 40 pM. In some embodiments, the binding affinityis any of about 10 nM, about 5 nM, about 1 nM, about 900 pM, about 800pM, bout 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM,about 200 pM, about 150 pM, about 100 pM, about 90 pM, about 80 pM,about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about10 pM. In still other embodiments, the binding affinity is about 2 pM,about 5 pM, about 10 pM, about 15 pM, about 20 pM, about 40 pM, orgreater than about 40 pM.

Methods of making antibodies and polypeptides are known in the art anddescribed herein.

Competition assays can be used to determine whether two antibodies bindthe same epitope by recognizing identical or sterically overlappingepitopes or one antibody competitively inhibits binding of anotherantibody to the antigen. These assays are known in the art. Typically,antigen is immobilized on a multi-well plate and the ability ofunlabeled antibodies to block the binding of labeled antibodies ismeasured. Common labels for such competition assays are radioactivelabels or enzyme labels.

Antibodies and polypeptides that specifically bind to Aβ can be screenedfor efficacy in removing amyloid deposit and other beneficial effects,such as improving cognition. For example, antibodies or polypeptides maybe administered to an animal having Alzheimer's pathology. Variousanimal models for Alzheimer's disease are known in the art. Followingadministration, level of compact and diffuse amyloid plaques, behavioranalysis for cognition, and microglia activation and microhemorrhage maytested using methods known in the art and described in detail in Example2. PCT WO 2004/032868; Wilcock et al., J. Neurosci. 23:3745-3751 (2003);Wilcock et al., J. Neuroinflammation 1:24 (2004).

Polynucleotides, Vectors and Host Cells

The invention also provides isolated polynucleotides encoding theantibodies and polypeptides of the invention (including an antibodycomprising the polypeptide sequences of the light chain and heavy chainvariable regions shown in FIG. 1), and vectors and host cells comprisingthe polynucleotide.

Accordingly, the invention provides polynucleotides (or compositions,including pharmaceutical compositions), comprising polynucleotidesencoding any of the following: (a) antibody 9TL or its variants shown inTable 3; (b) a fragment or a region of antibody 9TL or its variantsshown in Table 3; (c) a light chain of antibody 9TL or its variantsshown in Table 3; (d) a heavy chain of antibody 9TL or its variantsshown in Table 3; (e) one or more variable region(s) from a light chainand/or a heavy chain of antibody 9TL or its variants shown in Table 3;(f) one or more CDR(s) (one, two, three, four, five or six CDRs) ofantibody 9TL or its variants shown in Table 3; (g) CDR H3 from the heavychain of antibody 9TL; (h) CDR L3 from the light chain of antibody 9TLor its variants shown in Table 3; (i) three CDRs from the light chain ofantibody 9TL or its variants shown in Table 3; (j) three CDRs from theheavy chain of antibody 9TL or its variants shown in Table 3; (k) threeCDRs from the light chain and three CDRs from the heavy chain, ofantibody 9TL or its variants shown in Table 3; and (1) an antibodycomprising any one of (b) through (k). In some embodiments, thepolynucleotide comprises either or both of the polynucleotide(s) shownin SEQ ID NO:9 and SEQ ID NO:10.

In another aspect, the invention provides polynucleotides encoding anyof the antibodies (including antibody fragments) and polypeptidesdescribed herein, such as antibodies and polypeptides having impairedeffector function. Polynucleotides can be made by procedures known inthe art.

In another aspect, the invention provides compositions (such as apharmaceutical compositions) comprising any of the polynucleotides ofthe invention. In some embodiments, the composition comprises anexpression vector comprising a polynucleotide encoding the 9TL antibodyas described herein. In other embodiment, the composition comprises anexpression vector comprising a polynucleotide encoding any of theantibodies or polypeptides described herein. In still other embodiments,the composition comprises either or both of the polynucleotides shown inSEQ ID NO:9 and SEQ ID NO:10. Expression vectors, and administration ofpolynucleotide compositions are further described herein.

In another aspect, the invention provides a method of making any of thepolynucleotides described herein.

Polynucleotides complementary to any such sequences are also encompassedby the present invention. Polynucleotides may be single-stranded (codingor antisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules. RNA molecules include HnRNA molecules,which contain introns and correspond to a DNA molecule in a one-to-onemanner, and mRNA molecules, which do not contain introns. Additionalcoding or non-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an antibody or a portion thereof) or may comprisea variant of such a sequence. Polynucleotide variants contain one ormore substitutions, additions, deletions and/or insertions such that theimmunoreactivity of the encoded polypeptide is not diminished, relativeto a native immunoreactive molecule. The effect on the immunoreactivityof the encoded polypeptide may generally be assessed as describedherein. Variants preferably exhibit at least about 70% identity, morepreferably at least about 80% identity and most preferably at leastabout 90% identity to a polynucleotide sequence that encodes a nativeantibody or a portion thereof.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e. gaps) of 20 percent or less, usually 5 to 15 percent, or10 to 12 percent, as compared to the reference sequences (which does notcomprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e. the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Variants may also, or alternatively, be substantially homologous to anative gene, or a portion or complement thereof. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA sequence encoding a nativeantibody (or a complementary sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

The polynucleotides of this invention can be obtained using chemicalsynthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al. (1989).

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al. eds., Birkauswer Press, Boston (1994).

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., (1989), for example.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a polynucleotide according to the invention. It is impliedthat an expression vector must be replicable in the host cells either asepisomes or as an integral part of the chromosomal DNA. Suitableexpression vectors include but are not limited to plasmids, viralvectors, including adenoviruses, adeno-associated viruses, retroviruses,cosmids, and expression vector(s) disclosed in PCT Publication No. WO87/04462. Vector components may generally include, but are not limitedto, one or more of the following: a signal sequence; an origin ofreplication; one or more marker genes; suitable transcriptionalcontrolling elements (such as promoters, enhancers and terminator). Forexpression (i.e., translation), one or more translational controllingelements are also usually required, such as ribosome binding sites,translation initiation sites, and stop codons.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

The invention also provides host cells comprising any of thepolynucleotides described herein. Any host cells capable ofover-expressing heterologous DNAs can be used for the purpose ofisolating the genes encoding the antibody, polypeptide or protein ofinterest. Non-limiting examples of mammalian host cells include but notlimited to COS, HeLa, and CHO cells. See also PCT Publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotes (such asE. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; orK. lactis). Preferably, the host cells express the cDNAs at a level ofabout 5 fold higher, more preferably 10 fold higher, even morepreferably 20 fold higher than that of the corresponding endogenousantibody or protein of interest, if present, in the host cells.Screening the host cells for a specific binding to Aβ₁₋₄₀ is effected byan immunoassay or FACS. A cell overexpressing the antibody or protein ofinterest can be identified.

Diagnostic Uses of 9TL Derived Antibodies and Anti-Aβ Antibodies HavingImpaired Effector Function

Antibody 9TL which binds to C-terminus of Aβ₁₋₄₀ may be used to identifyor detect the presence or absence of Aβ₁₋₄₀. For simplicity, referencewill be made generally to 9TL or antibodies with the understanding thatthese methods apply to any of Aβ₁₋₄₀ binding embodiments (such aspolypeptides) described herein. Detection generally involves contactinga biological sample with an antibody described herein that binds toAβ₁₋₄₀ and the formation of a complex between Aβ₁₋₄₀ and an antibody(e.g., 9TL) which binds specifically to Aβ₁₋₄₀. The formation of such acomplex can be in vitro or in vivo. The term “detection” as used hereinincludes qualitative and/or quantitative detection (measuring levels)with or without reference to a control.

Any of a variety of known methods can be used for detection, including,but not limited to, immunoassay, using antibody that binds thepolypeptide, e.g. by enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA) and the like; and functional assay for theencoded polypeptide, e.g. binding activity or enzymatic assay. In someembodiments, the antibody is detectably labeled. Other embodiments areknown in the art and described herein.

Antibodies and polypeptides of the invention can be used in thedetection, diagnosis and monitoring of a disease, condition, or disorderassociated with altered or aberrant Aβ or βAPP expression, such asAlzheimer's disease and Down's syndrome. Thus, in some embodiments, theinvention provides methods comprises contacting a specimen (sample) ofan individual suspected of having altered or aberrant Aβ expression withan antibody or polypeptide of the invention and determining whether thelevel of Aβ₁₋₄₀ differs from that of a control or comparison specimen.In other embodiments, the invention provides methods comprisescontacting a specimen (sample) of an individual and determining level ofAβ₁₋₄₀ expression.

For diagnostic applications, the antibody may be labeled with adetectable moiety including but not limited to radioisotopes,fluorescent labels, and various enzyme-substrate labels. Methods ofconjugating labels to an antibody are known in the art. In otherembodiment of the invention, antibodies of the invention need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibodies of the invention.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The antibodies may also be used for in vivo diagnostic assays, such asin vivo imaging. Generally, the antibody is labeled with a radionuclide(such as ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, or ³H) so that the cells ortissue of interest can be localized using immunoscintiography.

The antibody may also be used as staining reagent in pathology,following techniques well known in the art.

Anti-Aβ antibodies having impaired effector function may be used formeasuring brain amyloid burden for diagnosis of subject at risk of ordiagnosed with AD, and assessing progress of any treatment and diseasestage. It has been reported that peripheral administration of amonoclonal anti-Aβ antibody results in a rapid increase in plasma Aβ andthe magnitude of this increase is highly correlated with amyloid burdenin the hippocampus and cortex. DeMattos et al., Science 295:2264-2267(2002). In some embodiments, an anti-Aβ antibody having impairedeffector function is administered to a subject, and level of Aβ in theplasma is measured, whereby an increase in plasma Aβ indicates presenceand/or level of brain amyloid burden in the subject. These methods maybe used to monitor effectiveness of the treatment and disease stage andto determine future dosing and frequency. Antibodies having impairedeffector function may have a better safety profile and provide advantagefor these diagnostic uses.

Methods of Using Anti-A/I Antibody for Therapeutic Purposes

The antibodies (including polypeptides), polynucleotides, andpharmaceutical compositions described herein can be used in methods fortreating, preventing and inhibiting the development of a diseasecharacterized by aberrant deposition of a protein in the brain of asubject. The methods comprise administering to the subject an effectiveamount of an antibody that specifically binds to the protein or theprotein deposit or a polynucleotide encoding the antibody, wherein theantibody has impaired effector function. For example, an antibody whichspecifically binds to prion protein or aggregated form of prion proteinand has impaired effector function may be administered to a subject forprophylactic and/or therapeutic treatment of Prion diseases; an antibodywhich specifically binds to synuclein (e.g., alpha-synuclein) oraggregated form of synuclein and has impaired effector function may beadministered to a subject for prophylactic and/or therapeutic treatmentof Parkinson's disease.

The antibodies (including polypeptides), polynucleotides, andpharmaceutical compositions described herein can be used in methods fortreating, preventing and inhibiting the development of Alzheimer'sdisease and other diseases associated with altered Aβ or βAPPexpression, or accumulation or deposit of Aβ peptide (collectivelytermed “Aβ-associated diseases”), such as Down's syndrome, Parkinson'sdisease, multi-infarct dementia, mild cognitive impairment, cerebralamyloid angiopathy, vascular disorder caused by deposit of Aβ peptide inblood vessels (such as stroke and HCHWA-D). Such methods compriseadministering the antibodies, polypeptides, or polynucleotides, or apharmaceutical composition to the subject. In prophylactic applications,pharmaceutical compositions or medicaments are administered to a patientsusceptible to, or otherwise at risk of, Alzheimer's disease (or otherAβ-associated disease) in an amount sufficient to eliminate or reducethe risk, lessen the severity, or delay the outset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. In therapeuticapplications, compositions or medicaments are administered to a patientsuspected of, or already suffering from such a disease in amountsufficient to cure, or at least partially arrest, the symptoms of thedisease (biochemical, histological and/or behavioral), including itscomplications and intermediate pathological phenotypes in development ofthe disease.

The invention also provides a method of delaying development of asymptom associated with Alzheimer's disease (or other Aβ-associateddisease) in a subject comprising administering an effective dosage of apharmaceutical composition comprising an antibody, a polypeptide, or apolynucleotide described herein to the subject. Symptoms associated withAlzheimer disease includes, but not limited to, abnormalities of memory,problem solving, language, calculation, visuospatial perception,judgment, and behavior.

This invention also provides methods of inhibiting or suppressing theformation of amyloid plaques and/or Aβ accumulation in a subjectcomprising administering an effective dose of a pharmaceuticalcomposition comprising an antibody, a polypeptide, or a polynucleotidedescribed herein described herein to the subject. In some embodiments,the amyloid plaques are in the brain of the subject. In someembodiments, the amyloid plaques are in the cerebral vasculature of thesubject. In other embodiments, the Aβ accumulation is in the circulatorysystem of the subject.

This invention also provides methods of reducing amyloid plaques and/orreducing or slowing Aβ accumulation in a subject comprisingadministering an effective dose of a pharmaceutical compositioncomprising an antibody, a polypeptide, or a polynucleotide describedherein to the subject. In some embodiments, the amyloid plaques are inthe brain of the subject. In some embodiments, the amyloid plaques arein the cerebral vasculature of the subject. In other embodiments, the Aβaccumulation is in the circulatory system of the subject.

This invention also provides methods of removing or clearing amyloidplaques and/or Aβ accumulation in a subject comprising administering aneffective dose of a pharmaceutical composition comprising an antibody, apolypeptide, or a polynucleotide described herein to the subject. Insome embodiments, the amyloid plaques are in the brain of the subject.In some embodiments, the amyloid plaques are in the cerebral vasculatureof the subject. In other embodiments, the Aβ accumulation is in thecirculatory system of the subject.

This invention also provides methods of reducing Aβ peptide in a tissue(such as brain), inhibiting and/or reducing accumulation of Aβ peptidein a tissue (such as brain), and inhibiting and/or reducing toxiceffects of Aβ peptide in a tissue (such as brain) in a subjectcomprising administering an effective dose of a pharmaceuticalcomposition comprising an antibody, a polypeptide, or a polynucleotidedescribed herein to the subject. Aβ polypeptide may be in soluble,oligomeric, or deposited form. Oligomeric form of Aβ may be composed of2-50 Aβ polypeptides, which can be a mixture of full length 1-40 and1-42 peptides and/or any truncated version of the these peptides.

The invention also provides methods of improving cognition or reversingcognitive decline associated with diseases associated with amyloiddeposit of Aβ in a subject, such as Alzheimer's disease, comprisingadministering an effective dosage of a pharmaceutical compositioncomprising an antibody, a polypeptide, or a polynucleotide describedherein to the subject.

The invention also provides methods for treating or preventing diseasesassociated with amyloid deposit of Aβ, comprising administering to thesubject an effective dosage of a pharmaceutical composition comprisingan antibody that specifically binds to a beta-amyloid peptide or anaggregated form of a beta-amyloid peptide, wherein the antibodycomprises an Fc region with a variation from a naturally occurring Fcregion, wherein the variation results in impaired effector function,whereby the administration of the antibody causes less cerebralmicrohemorrhage than administration of an antibody without thevariation.

Aberrant deposition of proteins in the brain is associated with a numberof disorders, some of which can also be characterized as amyloiddiseases or amyloidoses, due to concurrent deposition of amyloid-formingproteins.

Amyloidoses are disorders that are characterized by extracellulardeposition of protein fibrils, which form amyloid deposits. While themajority of these conditions are associated with amyloid deposition inthe periphery, there are a number of amyloidoses in which centralnervous system fibril deposition predominates. WO 00/72876 describes anumber of central and peripheral amyloidoses.

Alzheimer's disease is the most well-known and probably the most commonamyloid disease of the central nervous system. This condition ischaracterized by A-beta-containing plaques and neurofibrillatorytangles, as summarized elsewhere. Various forms of senile dementia arealso associated with similar, but less progressive, A-beta plaqueformation, as is Down's syndrome.

Aberrant deposition of endostatin (a 20 kDa C-terminal fragment ofcollagen XVIII) has been observed in brains of Alzheimer's patients,where it co-localizes with amyloid-beta(1-40). Deininger, M. H., et al.,(2003) J. Neurosci 22(24): 10621-10626.

A variant of the amyloidogenic protein cystatin C, L68Q cystatin C, isassociated with massive cerebral amyloidosis leading to brain hemorrhageand death in early adult life in a hereditary form of amyloid angiopathy(hereditary cystatin C amyloid angiopathy). A normal variant of cystatinC (wt¹ cystatin C) can be found associated with A-beta peptide as acomponent of amyloid plaques in Alzheimer's disease.

In studies to determine whether exogenous agents could suppressformation of dimers of cystatin C (either L68Q or wt¹ cystatin C),Nilsson and co-workers incubated antibodies directed to wt¹ cystatin Cwith monomeric forms of the two variant proteins in solution andobserved decreased dimerization of the proteins. Nilsson, M. et al.(2004) J. Biol. Chem. 279(3): 24236-45.

Down syndrome is characterized by A-beta peptide plaque deposition,apoptotic cell death and aberrant dendritic arborization, in part due toconstitutively increased expression of genes that include amyloidprecursor protein (APP) and other proteins (superoxide dismutase I, andS100-beta)—all located within the Down locus. There is also aberrantexpression of genes that are not linked to the Down locus (genes w/in asegment of chromosome 21)—GAP-43, nitric oxide synthase 3, neuronalthread protein, pro-apoptosis genes such as p53, Bax and LI-1beta-converting enzyme. Expression of these non-Down locus genescorrelates with proliferation of dystrophic neuritis and apoptotic celldeath. de la Monte, S. M. 1999., J. Neural Transm. Suppl. 57: 1-19.

Brain deposition of amyloid plaques formed from A-beta peptide is a alsoa common pathologic feature in HIV-AIDS patients. Green, D. A., et al.(2005) AIDS 19(4): 407-11. Similarly, deposition of A-betapeptide-containing plaques have also been observed shortly followingtraumatic brain injury in humans, where A-beta co-localizes with APP andneurofilament proteins in swollen axons. Smith, D. H., et al. (2003):98(5): 1072-7. Brains of patients with end-stage acquiredimmunodeficiency syndrome (AIDS) were also shown to have increasedlevels of ubiquitin-stained dotlike deposits (Ub-dots). Gelman, B. B.,and Schuenke, K. (2004) J. Neurovirol 10(2): 98-108.

Amyloidoma is a relatively rare form of CNS amyloidosis, presenting inthe form of an amyloid tumor, usually in the choroid plexus, withsecondary extensions into white matter. Primary amyloidomas of the brainparenchyma comprise lesions composed of amyloid AL lambda light chain.Tabatbai, G., et al. (2005) Arch Neurol. 62(3): 477-80.

Multifocal white matter lesions have been observed in brain MRIs inpatients carrying the gene(s) for the transthyretin Tyr77 (Tyr77 FAP)variant of familial amyloid polyneuropathy (FAP). Lossos, A., et al.Eur. Neurol. 2005. 53(2): 55-9.

Familial leptomeningeal amyloidosis is associated with a geneticabnormality of the transthyretin (TTR) variant Asp18Gly (D18G). Jin, K.,et al J. Neurol. Neurosurg. Psychiatry (2004) 75(10): 1463-66. The D18Gvariant form of TTR has been also shown to lead to CNS amyloidosis inHungarian patients. According to one report, small molecule stabilizersof a tetrameric form of the protein may prevent amyloidogenesis.Hammarstrom, P. et al. (2003) Biochemistry 42(22): 6656-63.

Mutations in genes encoding alpha-synuclein have been found to beresponsible for at least some familial forms of Parkinson's disease,where alpha-synuclein has been shown to be abnormally derivatized and toform neuronal and glial inclusions. Alpha synuclein also forms fibrilsin vitro, leading to the categorization of Parkinson's disease as abrain amyloidosis. Trojanowski, J. Q. and Lee, V. M. (2003) Ann NY Acad.Sci. 991: 107-110.

Alpha-synuclein inclusions in oligodendroglia characterize multiplesystem atrophy (MSA). Kahle, P. M., et al. 2002. EMBO rep. 3(6): 583-8.

PrP^(Sc) is aberrant form of cellular prion protein (PrP^(C)), acopper-binding glycoprotein attached to the cell membrane of neurons andother cells. PrP amyloid accumulation is commonly associated with PrPcerebral amyloid angiopathy (PrP-CAA), where the accumulation is inneurofibrillary tangles and vascular amyloid, and inGerstmann-Straussler-Scheinker disease, where parenchymal amyloidosismay be present in association with spongiform degeneration ofneurofibrillary tangles. Ghetti, B., et al. Clin. Lab. Med. (2003)23(1): 65-85. PrP^(Sc) deposition is also found in human spongiformencephalopathy (variant Creutzfeld-Jacob Disease). Paracrine inhibitionof prion propagation by anti-PrP single-chain Fv miniantibodies has beenreported. Heppner et al., J. Viol. 79:8330-8; 2005.

The following table provides a summary of examples of diseasesassociated with aberrant brain protein deposition and protein componentsfor the deposit. Antibody against these components may be generatedusing methods known in the art and methods described herein, orantibodies known in the art.

Condition Protein component(s) Alzheimer's disease A-beta peptide wt¹Cystatin C Endostatin Amyloidoma A-lambda light chain Cerebral amyloidangiopathy 1. A-beta peptide (CAA) 2. L68Q Cystatin C 3. PrP^(Sc)Spongiform encephalopathy PrP^(Sc) (variant Creutzfeld-Jacob Disease)Familial amyloid polyneuropathy Transthyretin variant Tyr77 AIDS 1.A-beta peptide 2. Ubiquitin Traumatic brain injury A-beta peptideFamilial leptomeningeal Transthyretin variant Asp18Gly amyloidosis(D18G) Parkinson's Disease Alpha synucleinGerstmann-Straussler-Scheinker PrP^(Sc) disease Down Syndrome A-betapeptide Superoxide dismutase S100-beta GAP-43 Nitric oxide synthase 3Neuronal thread protein P53 Bax LI-1 beta-converting enzyme MultipleSystem Atrophy Alpha-synuclein

The methods described herein (including prophylaxis or therapy) can beaccomplished by a single direct injection at a single time point ormultiple time points to a single or multiple sites. Administration canalso be nearly simultaneous to multiple sites. Frequency ofadministration may be determined and adjusted over the course oftherapy, and is base on accomplishing desired results. In some cases,sustained continuous release formulations of antibodies (includingpolypeptides), polynucleotides, and pharmaceutical compositions of theinvention may be appropriate. Various formulations and devices forachieving sustained release are known in the art.

Patients, subjects, or individuals include mammals, such as human,bovine, equine, canine, feline, porcine, and ovine animals. The subjectis preferably a human, and may or may not be afflicted with disease orpresently show symptoms. In the case of Alzheimer's disease, virtuallyanyone is at risk of suffering from Alzheimer's disease if he or shelives long enough. Therefore, the present methods can be administeredprophylactically to the general population without the need for anyassessment of the risk of the subject patient. The present methods areuseful for individuals who do have a known genetic risk of Alzheimer'sdisease. Such individuals include those having relatives who haveexperienced this disease, and those whose risk is determined by analysisof genetic or biochemical markers. Genetic markers of risk towardAlzheimer's disease include mutations in the APP gene, particularlymutations at position 717 and positions 670 and 671 referred to as theHardy and Swedish mutations respectively (see Hardy (1997) TrendsNeurosci. 20:154-9). Other markers of risk are mutations in thepresenilin genes, PS1 and PS2, and ApoE4, family history of AD,hypercholesterolemia or atherosclerosis. Individuals presently sufferingfrom Alzheimer's disease can be recognized from characteristic dementia,as well as the presence of risk factors described above. In addition, anumber of diagnostic tests are available for identifying individuals whohave AD. These include measurement of CSF tau and Aβ42 levels. Elevatedtau and decreased Aβ42 levels signify the presence of AD. Individualssuffering from Alzheimer's disease can also be diagnosed by ADRDA(Alzheimer's Disease and Related Disorders Association) criteria. Inasymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by variousways known in the art over time. In the case of potential Down'ssyndrome patients, treatment can begin antenatally by administeringtherapeutic agent to the mother or shortly after birth.

The pharmaceutical composition that can be used in the above methodsinclude, any of the antibodies, polypeptides, and/or polynucleotidesdescribed herein. In some embodiments, antibody is antibody 9TL or itsvariants shown in Table 3. In some embodiments, the antibody is anantibody that specifically binds to an Aβ peptide and comprises aconstant region having impaired effector function.

Administration and Dosage

The antibody is preferably administered to the mammal in a carrier;preferably a pharmaceutically-acceptable carrier. Suitable carriers andtheir formulations are described in Remington's Pharmaceutical Sciences,18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990;and Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing, 2000. Typically, an appropriate amount of apharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of the carrier include saline,Ringer's solution and dextrose solution. The pH of the solution ispreferably from about 5 to about 8, and more preferably from about 7 toabout 7.5. Further carriers include sustained release preparations suchas semipermeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the route of administration andconcentration of antibody being administered.

The antibody can be administered to the mammal by injection (e.g.,systemic, intravenous, intraperitoneal, subcutaneous, intramuscular,intraportal, intracerebral, intracerebralventricular, and intranasal),or by other methods, such as infusion, which ensure its delivery to thebloodstream in an effective form. The antibody may also be administeredby isolated perfusion techniques, such as isolated tissue perfusion, toexert local therapeutic effects. Intravenous injection is preferred.

Effective dosages and schedules for administering the antibody may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibody that must be administered will vary depending on, forexample, the mammal that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered to the mammal. Guidance in selecting appropriatedoses for antibody is found in the literature on therapeutic uses ofantibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al.,eds., Noges Publications, Park Ridge, N.J., 1985, ch. 22 and pp.303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haberet al., eds., Raven Press, New York, 1977, pp. 365-389. A typical dailydosage of the antibody used alone might range from about 1 μg/kg to upto 100 mg/kg of body weight or more per day, depending on the factorsmentioned above. Generally, any of the following doses may be used: adose of at least about 50 mg/kg body weight; at least about 10 mg/kgbody weight; at least about 3 mg/kg body weight; at least about 1 mg/kgbody weight; at least about 750 μg/kg body weight; at least about 500μg/kg body weight; at least about 250 ug/kg body weight; at least about100 μg/kg body weight; at least about 50 μg/kg body weight; at leastabout 10 ug/kg body weight; at least about 1 μg/kg body weight, or more,is administered. Antibodies may be administered at lower doses or lessfrequent at the beginning of the treatment to avoid potential sideeffect, such as temporary cerebral amyloid angiopathy (CAA).

In some embodiments, more than one antibody may be present. Suchcompositions may contain at least one, at least two, at least three, atleast four, at least five different antibodies (including polypeptides)of the invention.

The antibody may also be administered to the mammal in combination witheffective amounts of one or more other therapeutic agents. The antibodymay be administered sequentially or concurrently with the one or moreother therapeutic agents. The amounts of antibody and therapeutic agentdepend, for example, on what type of drugs are used, the pathologicalcondition being treated, and the scheduling and routes of administrationbut would generally be less than if each were used individually.

Following administration of antibody to the mammal, the mammal'sphysiological condition can be monitored in various ways well known tothe skilled practitioner.

The above principles of administration and dosage can be adapted forpolypeptides described herein.

A polynucleotide encoding an antibody or a polypeptide described hereinmay also be used for delivery and expression of the antibody or thepolypeptide in a desired cell. It is apparent that an expression vectorcan be used to direct expression of the antibody. The expression vectorcan be administered systemically, intraperitoneally, intravenously,intramuscularly, subcutaneously, intrathecally, intraventricularly,orally, enterally, parenterally, intranasally, dermally, or byinhalation. For example, administration of expression vectors includeslocal or systemic administration, including injection, oraladministration, particle gun or catheterized administration, and topicaladministration. One skilled in the art is familiar with administrationof expression vectors to obtain expression of an exogenous protein invivo. See, e.g., U.S. Pat. Nos. 6,436,908; 6,413,942; and 6,376,471.

Targeted delivery of therapeutic compositions comprising apolynucleotide encoding an antibody of the invention can also be used.Receptor-mediated DNA delivery techniques are described in, for example,Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., GeneTherapeutics: Methods And Applications Of Direct Gene Transfer (J. A.Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al.,J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci.(USA) (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.Therapeutic compositions containing a polynucleotide are administered ina range of about 100 ng to about 200 mg of DNA for local administrationin a gene therapy protocol. Concentration ranges of about 500 ng toabout 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, andabout 20 μg to about 100 μg of DNA can also be used during a genetherapy protocol. The therapeutic polynucleotides and polypeptides ofthe present invention can be delivered using gene delivery vehicles. Thegene delivery vehicle can be of viral or non-viral origin (seegenerally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human GeneTherapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; andKaplitt, Nature Genetics (1994) 6:148). Expression of such codingsequences can be induced using endogenous mammalian or heterologouspromoters. Expression of the coding sequence can be either constitutiveor regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740; 4,777,127; GB Patent No. 2,200,651; and EP 0 345242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semlikiforest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373;ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923;ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus(AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769;WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administrationof DNA linked to killed adenovirus as described in Curiel, Hum. GeneTher. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA(see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP 0 524 968. Additionalapproaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, andin Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

Kits

The invention also provides articles of manufacture and kits containingmaterials useful for treating pathological conditions described herein,such as Alzheimer's disease or other Aβ-associated diseases (such asDown's syndrome, Parkinson's disease, multi-infarct dementia, mildcognitive impairment, cerebral amyloid angiopathy, vascular disordercaused by deposit of Aβ peptide in blood vessels (such as stroke andHCHWA-D)), or detecting or purifying Aβ or βAPP. The article ofmanufacture comprises a container with a label. Suitable containersinclude, for example, bottles, vials, and test tubes. The containers maybe formed from a variety of materials such as glass or plastic. Thecontainer holds a composition having an active agent which is effectivefor treating pathological conditions or for detecting or purifying Aβ orβAPP. The active agent in the composition is an antibody and preferably,comprises monoclonal antibodies specific for Aβ or βAPP. In someembodiments, the active agent comprises antibody 9TL or any antibodiesor polypeptides derived from antibody 9TL. In some embodiments, theactive agent comprises an anti-Aβ antibody or polypeptide havingimpaired effector function. In some embodiments, the anti-Aβ antibody orpolypeptide comprises a heavy chain constant region, wherein theconstant region has impaired effector function. The label on thecontainer indicates that the composition is used for treatingpathological conditions such as Alzheimer's disease or detecting orpurifying Aβ or βAPP, and may also indicate directions for either invivo or in vitro use, such as those described above.

The invention also provides kits comprising any of the antibodies (suchas 9TL), polypeptides, polynucleotides described herein. In someembodiments, the kit of the invention comprises the container describedabove. In other embodiments, the kit of the invention comprises thecontainer described above and a second container comprising a buffer. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for performing anymethods described herein (such as methods for treating Alzheimer'sdisease, and methods for inhibiting or reducing accumulation of Aβpeptide in the brain). In kits to be used for detecting or purifying Aβor βAPP, the antibody is typically labeled with a detectable marker,such as, for example, a radioisotope, fluorescent compound,bioluminescent compound, a chemiluminescent compound, metal chelator orenzyme.

In some embodiments, the invention provides compositions (describedherein) for use in any of the methods described herein, whether in thecontext of use as a medicament and/or use for manufacture of amedicament.

The following examples are provided to illustrate, but not to limit, theinvention.

EXAMPLES Example 1 Binding Affinity Determination of Antibody 9TL andits Variants A. General Methods

The following general methods were used in this example.

Expression Vector Used in Clone Characterization

Expression of the Fab fragment of the antibodies was under control of anIPTG inducible lacZ promotor similar to that described in Barbas (2001)Phage display: a laboratory manual, Cold Spring Harbor, N.Y., ColdSpring Harbor Laboratory Press pg 2.10. Vector pComb3X), however,modifications included addition and expression of the followingadditional domains: the human Kappa light chain constant domain and theCHI constant domain of IgG2a human immunoglobulin, Ig gamma-2 chain Cregion, protein accession number P01859; Immunoglobulin kappa lightchain (homosapiens), protein accession number CAA09181.

Small Scale Fab Preparation

Small scale expression of Fabs in 96 wells plates was carried out asfollows. Starting from E. coli transformed with a Fab library, colonieswere picked to inoculate both a master plate (agar LB+Ampicillin (50μg/ml)+2% Glucose) and a working plate (2 ml/well, 96 well/platecontaining 1.5 mL of LB+Ampicillin (50 μg/ml)+2% Glucose). Both plateswere grown at 30° C. for 8-12 hours. The master plate was stored at 4°C. and the cells from the working plate were pelleted at 5000 rpm andresuspended with 1 mL of LB+Ampicillin (50 μg/ml)+1 mM IPTG to induceexpression of Fabs. Cells were harvested by centrifugation after 5 hexpression time at 30° C., then resuspended in 500 μL of buffer HBS-P(10 mM HEPES buffer pH 7.4, 150 mM NaCl, 0.005% P20). Lysis of HBS-Presuspended cells was attained by one cycle of freezing (−80° C.) thenthawing at 37° C. Cell lysates were centrifuged at 5000 rpm for 30 minto separate cell debris from supernatants containing Fabs. Thesupernatants were then injected into the BIAcore plasmon resonanceapparatus to obtain affinity information for each Fab. Clones expressingFabs were rescued from the master plate to sequence the DNA and forlarge scale Fab production and detailed characterization as describedbelow.

Large Scale Fab Preparation

To obtain detailed kinetic parameters, Fabs were expressed and purifiedfrom large cultures. Erlenmeyer flasks containing 200 mL ofLB+Ampicillin (50 μg/ml)+2% Glucose were inoculated with 5 mL of overnight culture from a selected Fab-expressing E. coli clone. Clones wereincubated at 30° C. until an OD_(550nm) of 1.0 was attained and theninduced by replacing the media for 200 ml, of LB+Ampicillin (50 μg/ml)+1mM IPTG. After 5 h expression time at 30° C., cells were pelleted bycentrifugation, then resuspended in 10 mL PBS (pH 8). Lysis of the cellswas obtained by two cycles of freeze/thaw (at −80° C. and 37° C.,respectively). Supernatant of the cell lysates were loaded onto Ni-NTAsuperflow sepharose (Qiagen, Valencia. CA) columns equilibrated withPBS, pH 8, then washed with 5 column volumes of PBS, pH 8. IndividualFabs eluted in different fractions with PBS (pH 8)+300 mM Imidazol.Fractions containing Fabs were pooled and dialized in PBS, thenquantified by ELISA prior to affinity characterization.

Full Antibody Preparation

For expression of full antibodies, heavy and light chain variableregions were cloned in mammalian expression vectors and transfectedusing lipofectamine into HEK 293 cells for transient expression.Antibodies were purified using protein A using standard methods.

Vector pDb.9TL.hFc2a is an expression vector comprising the heavy chainof the 9TL antibody, and is suitable for transient or stable expressionof the heavy chain. Vector pDb.9TL.hFc2a has nucleotide sequencescorresponding to the following regions: the murine cytomegaloviruspromoter region (nucleotides 1-612); a synthetic intron (nucleotides619-1507); the DHFR coding region (nucleotides 707-1267); human growthhormone signal peptide (nucleotides 1525-1602); heavy chain variableregion of 9TL (nucleotides 1603-1951); human heavy chain IgG2a constantregion containing the following mutations: A330P331 to S330S331 (aminoacid numbering with reference to the wildtype IgG2a sequence; see Eur.J. Immunol. (1999) 29:2613-2624); SV40 late polyadenylation signal(nucleotides 2960-3203); SV40 enhancer region (nucleotides 3204-3449);phage fl region (nucleotides 3537-4992) and beta lactamase (AmpR) codingregion (nucleotides 4429-5286). Vector pDb.9TL.hFc2a was deposited atthe ATCC on Jul. 20, 2004, and was assigned ATCC Accession No. PTA-6124.

Vector pEb.9TL.hK is an expression vector comprising the light chain ofthe 9TL antibody, and is suitable for transient expression of the lightchain. Vector pEb.9TL.hK has nucleotide sequences corresponding to thefollowing regions: the murine cytomegalovirus promoter region(nucleotides 1-612); human EF-1 intron (nucleotides 619-1142); humangrowth hormone signal peptide (nucleotides 1173-1150); antibody 9TLlight chain variable region (nucleotides 1251-1593); human kappa chainconstant region (nucleotides 1594-1914); SV40 late polyadenylationsignal (nucleotides 1932-2175); SV40 enhancer region (nucleotides2176-2421); phage fl region (nucleotides 2509-2964) and beta lactamase(AmpR) coding region (nucleotides 3401-4258). Vector pEb.9TL.hK wasdeposited at the ATCC on Jul. 20, 2004, and was assigned ATCC AccessionNo. PTA-6125.

Biacore Assay

Affinities of 9TL monoclonal antibody were determined using theBIAcore3000™ surface plasmon resonance (SPR) system (BIAcore, INC,Piscaway N.J.). One way of determining the affinity was immobilizing of9TL on CM5 chip and measuring binding kinetics of Aβ₁₋₄₀ peptide to theantibody. CM5 chips were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antibody 9TL or its variants was diluted into 10 mM sodium acetate pH4.0 or 5.0 and injected over the activated chip at a concentration of0.005 mg/mL. Using variable flow time across the individual chipchannels, a range of antibody density was achieved: 1000-2000 or2000-3000 response units (RU). The chip was blocked with ethanolamine.Regeneration studies showed that a solution containing 2 volumes ofPIERCE elution buffer and 1 volumes of 4 M NaCl effectively removed thebound Aβ₁₋₄₀ peptide while keeping the activity of 9TL on the chip forover 200 injections. HBS-EP buffer (0.01M HEPES, pH 7.4, 0.15 M NaCl, 3mM EDTA, 0.005% Surfactant P20) was used as running buffer for all theBIAcore assays. Serial dilutions (0.1-10× estimated K_(D)) of purifiedAβ₁₋₄₀ synthetic peptide samples were injected for 1 min at 100 μL/minand dissociation times of 10 min were allowed. Kinetic association rates(k_(on)) and dissociation rates (k_(off)) were obtained simultaneouslyby fitting the data to a 1:1 Langmuir binding model (Karlsson, R. Roos,H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-110)using the BIAevaluation program. Equilibrium dissociation constant(K_(D)) values were calculated as k_(off)/k_(on).

Alternatively, affinity was determined by immobilizing Aβ₁₋₄₀ peptide onSA chip and measuring binding kinetics of 9TL Fab and Fab of 9TLvariants to the immobilized Aβ₁₋₄₀ peptide. Affinities of 9TL Fabfragment and its variants Fab fragments were determined by SurfacePlasmon Resonance (SPR) system (BIAcore 3000™, BIAcore, Inc., Piscaway,N.J.). SA chips (streptavidin) were used according to the supplier'sinstructions. Biotinylated Aβ peptide 1-40 was diluted into HBS-EP (10mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20) and injected overthe chip at a concentration of 0.005 mg/mL. Using variable flow timeacross the individual chip channels, two ranges of antigen density wereachieved: 10-200 response units (RU) for detailed kinetic studies and500-600 RU for concentration studies and screening. Regeneration studiesshowed that 100 mM phosphoric acid (may also be followed by a solutioncontaining 2 volumes of 50 mM NaOH and 1 volume of 70% ethanol)effectively removed the bound Fab while keeping the activity of Aβpeptide on the chip for over 200 injections. HBS-EP buffer was used asrunning buffer for all the BIAcore assays. Serial dilutions (0.1-10×estimated KD) of purified Fab samples were injected for 2 min at 100μL/min and dissociation times of 10 min were allowed. The concentrationsof the Fab proteins were determined by ELISA and/or SDS-PAGEelectrophoresis using a standard Fab of known concentration (determinedby amino acid analysis). Kinetic association rates (k_(on)) anddissociation rates (k_(off)) were obtained simultaneously by fitting thedata to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam,L. Petersson, B. (1994). Methods Enzymology 6. 99-110) using theBIAevaluation program. Equilibrium dissociation constant (K_(D)) valueswere calculated as k_(off)/k_(on).

B. Binding Affinity of Antibody 9TL and its Variants to 41-40

The amino acid sequences of the heavy chain and light chain variableregions of antibody 9TL is shown in FIG. 1. The binding affinity of 9TLantibody to Aβ₁₋₄₀ determined using both methods of Biacore describedabove is shown in Table 2 below.

TABLE 2 Binding affinity of antibody 9TL and Fab fragment k_(on) (1/Ms)K_(off) (1/s) K_(D) (nM) 9TL mAb on CM5 chip, Aβ₁₋₄₀ 4.25 × 10⁵ 3.89 ×10⁻⁴ 0.9 flowed onto it Aβ₁₋₄₀ on SA chip, 9TL Fab 3.18 × 10⁵ 3.59 ×10⁻⁴ 1.13 flowed onto it

The amino acid sequence of the variants of 9TL is shown in Table 3below. All amino acid substitutions of the variants shown in Table 3 aredescribed relative to the sequence of 9TL. The binding affinity of Fabfragment of 9TL variants are also shown in Table 3. K_(D) and otherkinetic parameters were determined by BIAcore analysis described abovewith Aβ₁₋₄₀ immobilized on SA chip.

TABLE 3 Amino acid sequences and kinetic data for antibody 9TL variants.H1 k_(on) k_(off) K_(D) Clone (1) H2 H3 L1 L2 L3 (Ms⁻¹) (2) (s⁻¹) (nM)(3) 9TL 3.18 × 10⁵ 3.59 × 10⁻⁴ 1.13 22-T/I L102I 3.18 × 10⁵ 4.60 × 10⁻⁴1.45 C6 new L102T 3.56 × 10⁵ 9.20 × 10⁻⁴ 2.58 W1 Y31A, L102T 3.18 × 10⁵9.00 × 10⁻³ 28.30 A34S W8 Y31H, L102T 3.18 × 10⁵ 3.80 × 10⁻³ 11.95 A34S,K35A W5 Y31H, L102T 3.18 × 10⁵ 4.00 × 10⁻³ 12.58 K35A M1 L94M 3.18 × 10⁵8.60 × 10⁻⁴ 2.70 M2 L94N 3.18 × 10⁵ 1.10 × 10⁻³ 3.46 M3 L94C 3.18 × 10⁵1.30 × 10⁻³ 4.09 M4 L94F 3.18 × 10⁵ 9.95 × 10⁻⁴ 3.13 M5 L94V 3.18 × 10⁵1.65 × 10⁻³ 5.19 M6 L94K 3.18 × 10⁵ 4.10 × 10⁻³ 12.89 M7 L94S 3.18 × 10⁵6.00 × 10⁻³ 18.87 M8 L94Q 3.18 × 10⁵ 6.80 × 10⁻³ 21.38 M9 L94G 3.18 ×10⁵ 7.80 × 10⁻³ 24.53 M10 L94S 3.18 × 10⁵ 8.30 × 10⁻³ 26.10 M11 G96S3.18 × 10⁵ 2.00 × 10⁻³ 6.29 M12 G96T 3.18 × 10⁵ 3.30 × 10⁻³ 10.38 M13T97S 3.18 × 10⁵ 3.90 × 10⁻⁴ 1.23 M14 H98L 3.18 × 10⁵ 1.60 × 10⁻³ 5.03M15 Y99P 3.18 × 10⁵ 6.70 × 10⁻⁴ 2.11 M16 Y99A 3.18 × 10⁵ 7.00 × 10⁻⁴2.20 M17 Y99W 3.18 × 10⁵ 1.00 × 10⁻³ 3.14 M18 Y99Q 3.18 × 10⁵ 1.50 ×10⁻³ 4.72 M19 Y99M 3.18 × 10⁵ 1.70 × 10⁻³ 5.35 M20 Y99S 3.18 × 10⁵ 2.00× 10⁻³ 6.29 M21 Y99E 3.18 × 10⁵ 5.00 × 10⁻³ 15.72 M22 V101L 3.18 × 10⁵4.00 × 10⁻³ 12.58 M23 V101K 3.18 × 10⁵ 5.00 × 10⁻³ 15.72 M24 V101H 3.18× 10⁵ 6.00 × 10⁻³ 18.87 M25 V101T 3.18 × 10⁵ 8.00 × 10⁻³ 25.16 M26 V101A3.18 × 10⁵ 9.00 × 10⁻³ 28.30 M27 V101E 3.18 × 10⁵ 1.20 × 10⁻² 37.74 M28V101M 3.18 × 10⁵ 1.40 × 10⁻² 44.03 M29 L102S 3.18 × 10⁵ 7.60 × 10⁻⁴ 2.39M30 L102V 3.18 × 10⁵ 6.80 × 10⁻⁴ 2.14 M31 L99V 3.18 × 10⁵ 1.00 × 10⁻²31.45 M32 L99I 3.18 × 10⁵ 2.00 × 10⁻² 62.89 M33 Y100W 3.18 × 10⁵ 6.30 ×10⁻⁴ 1.98 M34 S101T 3.18 × 10⁵ 8.00 × 10⁻⁴ 2.52 M35 S101G 3.18 × 10⁵9.00 × 10⁻³ 28.30 M36 L102R 3.18 × 10⁵ 9.00 × 10⁻⁴ 2.83 M37 L102A 3.18 ×10⁵ 9.20 × 10⁻⁴ 2.89 M38 L102V 3.18 × 10⁵ 1.50 × 10⁻³ 4.72 M39 L102S3.18 × 10⁵ 2.30 × 10⁻³ 7.23 M40 L102T 3.18 × 10⁵ 4.50 × 10⁻³ 14.15 M41L102Q 3.18 × 10⁵ 1.00 × 10⁻² 31.45 M42 L102E 3.18 × 10⁵ 1.50 × 10⁻²47.17 M43 V104I 3.18 × 10⁵ 3.00 × 10⁻⁴ 0.94 M44 V104T 3.18 × 10⁵ 3.00 ×10⁻³ 9.43 M45 V104P 3.18 × 10⁵ 1.50 × 10⁻² 47.17 M46 V104C 3.18 × 10⁵2.00 × 10⁻² 62.89 M47 V104Q 3.18 × 10⁵ 2.00 × 10⁻² 62.89 M48 V104S 3.18× 10⁵ 2.60 × 10⁻² 81.76 M49 V104N 3.18 × 10⁵ 2.60 × 10⁻² 81.76 M50 V104F3.18 × 10⁵ 2.70 × 10⁻² 84.91 M51 Y105H 3.18 × 10⁵ 8.60 × 10⁻⁴ 2.70 M52Y105F 3.18 × 10⁵ 1.30 × 10⁻³ 4.09 M53 Y105W 3.18 × 10⁵ 1.30 × 10⁻³ 4.09M54 Y105S 3.18 × 10⁵ 2.40 × 10⁻³ 7.55 M55 Y105I 3.18 × 10⁵ 3.00 × 10⁻³9.43 M56 Y105V 3.18 × 10⁵ 3.50 × 10⁻³ 11.01 M57 Y105A 3.18 × 10⁵ 3.90 ×10⁻³ 12.26 (1) = All CDRs are extended CDRs including both Kabat andChothia CDRs. Amino acid residues are numbered sequentially. (2) =underlined k_(on) were experimentally determined. Others were estimatedto be the same as 9TL. (3) = K_(D) values were calculated as K_(D) =k_(off)/k_(on).

Example 2 Characterization of Epitope on Aβ1-40 Peptide that Antibody9TL Binds

To determine the epitope on Aβ polypeptide that is recognized byantibody 9TL, Surface Plasmon Resonance (SPR, Biacore 3000) bindinganalysis was used. Aβ₁₋₄₀ polypeptide coupled to biotin (Global PeptideServices, CO) was immobilized on a streptavidin-coated chip (SA chip).The binding of Aβ antibodies Fab fragments (at 50 nM) to the immobilizedAβ₁₋₄₀ in the absence or presence of different soluble fragments of theAβ peptide (at 10 pM, from American Peptide Company Inc., CA). Aminoacid sequences of Aβ₁₋₄₀, Aβ₁₋₄₂, and Aβ₁₋₄₃ are shown in below in Table4. The Aβ peptides which displaced binding of antibody 9TL Fab fragmentto Aβ₁₋₄₀ were Aβ₂₈₋₄₀, Aβ₁₋₄₀, Aβ₃₃₋₄₀, and Aβ₁₇₋₄₀, respectively (FIG.2). Thus, antibody 9TL binds to a C-terminal peptide (33-40) of Aβ₁₋₄₀.As shown in FIG. 2, the Aβ₁₋₂₈, Aβ₂₈₋₄₂, Aβ₂₂₋₃₅, Aβ₁₋₁₆, Aβ₁₋₄₃, andAβ₁₋₃₈ peptide did not inhibit the binding of antibody 9TL Fab fragment,suggesting that antibody 9TL binds to the C-terminus of Aβ₁₋₄₀ peptide.

In addition, Aβ₂₈₋₄₂ and Aβ₁₋₄₃ peptide did not inhibit binding ofantibody 9TL to Aβ₁₋₄₀ although they could readily inhibit Aβ₁₋₄₀binding to control antibody (antibody 2289, this antibody is describedin U.S. Appl. Pub. No. 2004/0146512 and WO04/032868) which bind to 16-28of Aβ₁₋₄₀. These results show that antibody 9TL preferentially binds toAβ₁₋₄₀, but not to Aβ₁₋₄₂ and Aβ₁₋₄₃.

TABLE 4 Amino acid sequences of beta amyloid peptides 1-40 (WT)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGVV (SEQ ID NO: 15) 1-42 (WT)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGVVIA (SEQ ID NO: 16) 1-43 (WT)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGVVIAT (SEQ ID NO: 17)

Example 3 Generation of Monoclonal Antibody 2H6 and Deglycosylated 2H6A. Generation and Characterization of Monoclonal Antibody 2H6

Mice were immunized with 25-100 μg of a peptide (amino acid 28-40 ofAβ₁₋₄₀) conjugated to KLH in adjuvant (50 μA per footpad, 100 μA totalper mouse) at about 16 consecutive week intervals as described inGeerligs H J et al., 1989, J. Immunol. Methods 124:95-102; Kenney J S etal., 1989, J. Immunol. Methods 121:157-166; and Wicher K et al., 1989,Int. Arch. Allergy Appl. Immunol. 89:128-135. Mice were first immunizedwith 50 μg of the peptide in CFA (complete Freud's adjuvant). After 21days, mice were secondly immunized with 25 μg of the peptide in IFA(incomplete Freud's adjuvant). Twenty three days later after the secondimmunization, third immunization was performed with 25 μg of the peptidein IFA. Ten days later, antibody titers were tested using ELISA. Forthimmunization was performed with 25 μg of the peptide in IFA 34 daysafter the third immunization. Final booster was performed with 100 μgsoluble peptide 32 days after the forth immunization.

Splenocytes were obtained from the immunized mouse and fused with NSOmyeloma cells at a ratio of 10:1, with polyethylene glycol 1500. Thehybrids were plated out into 96-well plates in DMEM containing 20% horseserum and 2-oxaloacetate/pyruvate/insulin (Sigma), andhypoxanthine/aminopterin/thymidine selection was begun. On day 8, 100 μlof DMEM containing 20% horse serum was added to all the wells.Supernatants of the hybrids were screened by using antibody captureimmunoassay. Determination of antibody class was done withclass-specific second antibodies.

A panel of monoclonal antibody-producing cell lines was selected forcharacterization. One cell line selected produces as antibody designated2H6. This antibody was determined to have IgG2b heavy chain.

The affinity of antibody 2H6 to Aβ₁₋₄₀ was determined. Monoclonalantibody 2H6 was purified from supernatants of hybridoma cultures usingprotein A affinity chromatography. The supernatants was equilibrated topH 8. The supernatants were then loaded to the protein A columnMabSelect (Amersham Biosciences #17-5199-02) equilibrated with PBS to pH8. The column was washed with 5 column volumes of PBS, pH 8. Theantibody was eluted with 50 mM citrate-phosphate buffer, pH 3. Theeluted antibody was neutralized with 1M Phosphate Buffer, pH 8. Thepurified antibody was dialyzed with PBS. The antibody concentration wasdetermined by SDS-PAGE, using a murine mAb standard curve.

2H6Fabs were prepared by papain proteolysis of the 2H6 full antibodyusing Immunopure Fab kit (pierce #44885) and purified by flow throughprotein A chromatography following manufacturer instructions.Concentration was determined by SDS-PAGE and A280 using 1 OD=0.6 mg/ml.

Affinities of 2H6 monoclonal antibody were determined using theBIAcore3000™ surface plasmon resonance (SPR) system (BIAcore, INC,Piscaway N.J.). One way of determining the affinity was immobilizing 2H6antibody on CM5 chip and measuring binding kinetics of Aβ₁₋₄₀ peptide tothe antibody. CM5 chips were activated withN-ethyl-N′(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions. 2H6monoclonal antibody was diluted into 10 mM sodium acetate pH 4.0 or 5.0and injected over the activated chip at a concentration of 0.005 mg/mL.Using variable flow time across the individual chip channels, a range ofantibody density was achieved: 1000-2000 or 2000-3000 response units(RU). The chip was blocked with ethanolamine. Regeneration studiesshowed that a mixture of Pierce elution buffer (Product No. 21004,Pierce Biotechnology, Rockford, Ill.) and 4 M NaCl (2:1) effectivelyremoved the bound Aβ₁₋₄₀ peptide while keeping the activity of 2H6antibody on the chip for over 200 injections. HBS-EP buffer (0.01MHEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20) was usedas running buffer for all the BIAcore assays. Serial dilutions (0.1-10×estimated K_(D)) of purified Aβ₁₋₄₀ synthetic peptide samples wereinjected for 1 min at 100 μL/min and dissociation times of 10 min wereallowed. Kinetic association rates (k_(on)) and dissociation rates(k_(off)) were obtained simultaneously by fitting the data to a 1:1Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson,B. (1994). Methods Enzymology 6. 99-110) using the BIAevaluationprogram. Equilibrium dissociation constant (K_(D)) values werecalculated as k_(off)/k_(on).

Alternatively, affinity was determined by immobilizing Aβ₁₋₄₀ peptide onSA chip and measuring binding kinetics of 2H6Fab to the immobilizedAβ₁₋₄₀ peptide. Affinities of 2H6Fab fragment was determined by SurfacePlasmon Resonance (SPR) system (BIAcore 3000™, BIAcore, Inc., Piscaway,N.J.). SA chips (streptavidin) were used according to the supplier'sinstructions. Biotinylated Aβ peptide 1-40 (SEQ ID NO:15) was dilutedinto HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20) andinjected over the chip at a concentration of 0.005 mg/mL. Using variableflow time across the individual chip channels, two ranges of antigendensity were achieved: 10-200 response units (RU) for detailed kineticstudies and 500-600 RU for concentration studies. Regeneration studiesshowed that a mixture of Pierce elution buffer and 4 M NaCl (2:1)effectively removed the bound Fab while keeping the activity of Aβpeptide on the chip for over 200 injections. HBS-EP buffer was used asrunning buffer for all the BIAcore assays. Serial dilutions (0.1-10×estimated 1(D) of purified Fab samples were injected for 2 min at 100μL/min and dissociation times of 10 min were allowed. The concentrationsof the Fab proteins were determined by ELISA and/or SDS-PAGEelectrophoresis using a standard Fab of known concentration (determinedby amino acid analysis). Kinetic association rates (k_(on)) anddissociation rates (k_(off)) were obtained simultaneously by fitting thedata to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam,L. Petersson, B. (1994). Methods Enzymology 6. 99-110) using theBIAevaluation program. Equilibrium dissociation constant (K_(D)) valueswere calculated as k_(off)/k_(on). The affinity of 2H6 antibodydetermined using both methods described above is shown in Table 5 below.

Affinity for murine antibody 2286, which binds to a peptide of aminoacid 28-40 of Aβ₁₋₄₀, was tested as described above. Antibody 2286 isdescribed in U.S. application Ser. No. 10/683,815 and PCT/US03/32080.

TABLE 5 Binding affinity of antibody 2H6 and 2286 k_(on) (1/Ms) K_(off)(1/s) K_(D) (nM) 2H6 mAb on CM5 chip, Aβ₁₋₄₀ 4.67 × 10⁵ 3.9 × 10⁻³ 9flowed on Aβ₁₋₄₀0 on SA chip, 2H6 Fab  6.3 × 10⁵ 3.0 × 10⁻³ 4.7 flowedon 2286 mAb on CM5 chip, Aβ₁₋₄₀ 1.56 × 10⁵ 0.0419 269 flowed on Aβ₁₋₄₀on SA chip, 2286 Fab  1.8 × 10⁵ 0.044  245 flowed on

To determine the epitope on Aβ polypeptide recognized by antibody 2H6,Surface Plasmon Resonance (SPR, Biacore 3000) binding analysis was used.Aβ₁₋₄₀ polypeptide (SEQ ID NO:15) coupled to biotin (Global PeptideServices, CO) was immobilized on a streptavidin-coated chip (SA chip).The binding of Aβ antibodies (at 100 nM) to the immobilized Aβ₁₋₄₀ inthe absence or presence of different soluble fragments of the Aβ peptide(at 16 μM, from American Peptide Company Inc., CA). The Aβ peptideswhich displaced binding of antibody 2H6 to Aβ₁₋₄₀ were Aβ₁₇₋₄₀, Aβ₃₃₋₄₀,and Aβ₁₋₄₀, respectively (FIG. 3). Thus, antibody 2H6 binds to aC-terminal peptide (33-40) of Aβ₁₋₄₀. However, this C-terminal peptide(33-40) of Aβ₁₋₄₀ did not displace binding of antibody 2286 to Aβ₁₋₄₀ atthe concentration tested. As shown in FIG. 3, the Aβ₁₋₃₈ peptide did notinhibit the binding of antibody 2H6 or antibody 2286 to Aβ₁₋₄₀,suggesting that, similar to antibody 2286, the epitope that antibody 2H6binds includes amino acids 39 and/or 40 of the Aβ₁₋₄₀ peptide (FIG. 3).

In addition, Aβ₁₋₄₂ and Aβ₁₋₄₃ peptide did not inhibit binding ofantibody 2H6 to Aβ₁₋₄₀ although they could readily inhibit Aβ₁₋₄₀binding to control antibody (antibody 2289, this antibody is describedin U.S. application Ser. No. 10/683,815 and PCT/US03/32080) which bindto 16-28 of Aβ₁₋₄₀ (FIG. 3). These results show that antibody 2H6preferentially binds to Aβ₁₋₄₀, but not to Aβ₁₋₄₂ and Aβ₁₋₄₃.

To further assess the involvement of discrete amino acid residues of theβ-amyloid peptide that antibody 2H6 binds, different Aβ₁₋₄₀ variants, inwhich each of the last 6 amino acids (Aβ₁₋₄₀ amino acid residues 35-40)was individually replaced by an alanine (alanine scanning mutagenesis),were generated by site directed mutagenesis. These Aβ₁₋₄₀ variants(sequences shown in Table 6) were expressed in E. coli asGlutathione-S-Transferase (GST) fusion proteins (Amersham PharmaciaBiotech, Piscataway, N.J. USA) followed by affinity purification on aGlutathione-Agarose beads (Sigma-Aldrich Corp., St. Louis, Mo., USA). Ascontrol, Wild-type (WT) Aβ₁₋₄₀ as well as Aβ₁₋₄₁, Aβ₁₋₄₂, and Aβ₁₋₃₉were also expressed as GST fusion proteins. Aβ₁₋₄₀, Aβ₁₋₄₁, Aβ₁₋₄₂,Aβ₁₋₃₉, as well as the six different variants (M35A(1-40), V36A(1-40),G37A(1-40), G38A(1-40), V39A(1-40), V40A(1-40) shown in Table 6) werethen immobilized (100 μl of 0.025 μg/μl of GST-peptide per well) ontoELISA assay plates and incubated with either of mAb 2286, 2289, and 2H6in serial dilution from 0.3 nM down (data using 0.3 nM mAb are shown inFIG. 4). After 10 consecutive washes, assay plates were incubated with100 μl of 0.03 μg/ml per well of Biotin-conjugated Goat-anti-Mouse (H+L)antibody (Vector Laboratories, vector #BA-9200, Burllingame Calif., USA)followed by 100 μl of 0.025 μg/ml per well of HRP-conjugatedStreptavidin (Amersham Biosciences Corp., #RPN4401V, NJ, USA). Theabsorbance of the plate was read at 450 nm.

TABLE 6 Amino acid sequences of beta amyloid peptides and variants1-40 (WT) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL (SEQ ID NO: 15) MVGGVV1-42 (WT) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL (SEQ ID NO: 16) MVGGVVIA1-43 (WT) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL (SEQ ID NO: 17) MVGGVVIAT1-41 (WT) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL (SEQ ID NO: 18) MVGGVVI1-39 (WT) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL (SEQ ID NO: 19) MVGGVM35A(1-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL (SEQ ID NO: 20) AVGGVVV36A(1-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL (SEQ ID NO: 21) MAGGVVG37A(1-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL (SEQ ID NO: 22) MVAGVVG38A(1-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL (SEQ ID NO: 23) MVGAVVV39A(1-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL (SEQ ID NO: 24) MVGGAVV40A(1-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL (SEQ ID NO: 25) MVGGVA

As shown in FIG. 4, Mab 2289 which was directed to amino acid 16 to 28of Aβ, recognized all variants with the same intensity and served asinternal positive control of protein concentration and protein integrityon the plate. Antibody 2H6 did not recognize Aβ₁₋₄₁, Aβ₁₋₃₉, or Aβ₁₋₄₂as shown in FIG. 4. Aβ₁₋₄₀ variants V40A, V39A, G38A, G37A, V36A, andM35A showed reduced binding to antibody 2H6, demonstrating that antibody2H6 epitope extended for at least 6 amino acids at the C terminal end ofAβ₁₋₄₀. Mutations of V and G to A are very conservative and are notlikely to produce important conformational changes in proteins,therefore, the large effect of these mutations to antibody 2H6 bindingmight be due to the ability of the antibody to differentiate between thementioned amino acids in the context of Aβ and these data demonstrated avery high degree of specificity for this antibody.

To determine whether 2H6 and 9TL compete for binding to Aβ₁₋₄₀,competition experiments were performed using Biacore assay. Antibody2H6, 9TL and 2289 were immobilized on different channels of a CM5 chip.CM5 chip channels were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antibody 2H6, 9TL, and 2289 were each diluted into 10 mM sodium acetatepH 4.0 and injected over an activated chip at a concentration of 0.005mg/mL. Antibody density was 1625 response units (RU) for 2H6; 4000 RUfor 9TL; and 2200 RU for 2289. Each channel was blocked withethanolamine. Aβ₁₋₄₀ peptide (150 uM) was flowed onto the chip for 2min. Then antibody 2H6 (to be tested for competition of binding) at 0.6uM was flowed onto the chip for 1 min. HBS-EP buffer (0.01M HEPES, pH7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20) was used as runningbuffer for all the BIAcore assays. After measuring binding of Aβ₁₋₄₀,all channels of the chip were regenerated by washing twice with amixture of Pierce elution buffer (Product No. 21004, PierceBiotechnology, Rockford, Ill.) and 4 M NaCl (2:1) for 6 sec. Competitionbinding was then performed for antibody 9TL, and then antibody 2289.Competition between 9TL and 2H6 for binding to Aβ₁₋₄₀ was observed, butno competition was observed between 9TL and 2289 or between 2H6 and2289. Observations of competition between the antibody immobilized andthe same antibody flowed onto the chip served as the positive control.

B. Antibody 2H6 Does not Bind to APP

To determine whether 2H6 binds to amyloid precursor proteins (APP),binding of 2H6 to cells transfected with wildtype APP was determined.293 cells were transfected with a cDNA encoding wild type human amyloidprecursor protein. Forty eight hours after the transfection, cells wereincubated on ice for 45 minutes with monoclonal antibodies anti-Aβ₁₋₄₆,anti-Aβ₁₆₋₂₈, or 2H6 (5 ug/ml in DMEM with 10% FCS). The cells were thenwashed three times in PBS for 5 minutes, fixed with 4% PFA. The cellswere washed three times again in PBS, and antibody binding was detectedwith secondary Cy3-conjugated goat anti-mouse antibody (dilution of1:500) from Jackson Immunoresearch under fluorescence microscope.

Anti-Aβ₁₋₄₆ and anti-Aβ₁₆₋₂₈ antibodies, which recognize N-terminal orcentral epitopes in Aβ, both showed significant binding to APP precursorproteins expressed on cells. In contrast, 2H6 did not bind to APPexpressing cells.

C. Generation of Deglycosylated Antibody 2H6

To generate deglycosylated antibody 2H6, purified antibody 2H6 wasincubated at 37° C. for 7 days with peptide-N-glycosidase F (Prozyme,0.05 Upper mg of antibody) in 20 mM Tris-HCl pH 8.0. Completeness ofdeglycosylation was verified by MALDI-TOF-MS and protein gelelectrophoresis. Deglycosylated antibodies were purified by Protein Achromatography and endotoxin was removed by Q-Sepharose. The bindingaffinity to Aβ₁₋₄₀ of the deglycosylated 2H6 was tested using Biacoreassay described above, and the binding affinity of the deglycosylated2H6 to Aβ₁₋₄₀ was found to be identical to the intact antibody 2H6.

Example 4 Reversal of Cognitive Deficits and Histological Symptoms withLess Microhemorrhage in an Animal Model of Alzheimer's Disease byAdministration of Deglycosylated 2H6 A. Experimental Protocol

Administration of Antibodies.

Transgenic mice over-expressing the “Swedish” mutant amyloid precursorprotein (APP Tg2576 with K670N/M671; Hsiao et al., Science 274:99-102(1996)) were used for the experiments. The Alzheimer's-like phenotypepresent in these mice has been well-characterized. Holcomb et al., Nat.Med. 4:97-100 (1998); Holcomb et al., Behay. Gen. 29:177-185 (1999); andMcGowan E, Neurobiol. Dis. 6:231-244 (1999). For the sixteen weekstreatment study, APP-transgenic mice, aged 20 months, were assigned toone of the four groups. The first group received weekly intraperitonealanti-Aβ antibody 2H6 (mouse monoclonal anti-human Aβ₂₈₋₄₀ IgG2bdescribed in Example 3) injections for a period of 16 weeks (n=4). Thesecond group received weekly intraperitoneal deglycosylated anti-Aβantibody 2H6 (produced as described in Example 3) injections for aperiod of 16 weeks (n=5). The third group received weeklyintraperitoneal anti-AMN antibody (2906; mouse-monoclonalanti-Drosophila amnesiac protein IgG1) injections for a period of 16weeks (n=6). Non-transgenic littermates were treated for 16 weeks witheither anti-AMN antibody (n=4) or 2H6 (n=2).

Behavioral Analysis.

Following 16 weeks of antibody treatment, the mice from the study weresubjected to a two-day radial-arm water-maze paradigm as describedpreviously. Wilcock et al., J. Neuroinflammation 1:24 (2004). Theapparatus was a 6-arm maze as described previously. Gordon et al.,Neurobiol. Aging 22:377-385 (2001). On day one, 15 trials were run inthree blocks of 5. A cohort of 4 mice were run sequentially for eachblock (i.e., each of 4 mice get trial one, then the same mice get trialtwo, etc.). After each 5-trial block, a second cohort of mice was runpermitting an extended rest period before mice were exposed to thesecond block of 5 trials. The goal arm was different for each mouse in acohort to minimize odor cues. The start arm was varied for each trial,with the goal arm remaining constant for a given individual for bothdays. For the first 11 trials, the platform was alternately visible thenhidden (hidden for the last 4 trials). On day two, the mice were run inexactly the same manner as day one except that the platform was hiddenfor all trials. The number of errors (incorrect arm entries) wasmeasured in a one-minute time frame. Mice failing to make an arm choicein 20 seconds were assigned one error, but no mice in this study had tobe assigned an error in this manner. Due to the numbers of mice in thestudy, the tester was unaware of treatment group identity of each mouse.Since the dependent measures in the radial-arm water-maze task werequantitative, not evaluative, the potential for tester bias was reduced.In order to minimize the influence of individual trial variability, eachmouse's errors for 3 consecutive trials were averaged producing 5 datapoints for each day, which were analyzed statistically by ANOVA usingStatView (SAS Institute Inc., NC).

Histological Analysis.

On the day of sacrifice, mice were weighed, overdosed with 100 mg/kgNembutal (Abbott laboratories, North Chicago, Ill.), and thenintracardially perfused with 25 mL of 0.9% sodium chloride. Brains wererapidly removed, and the left half of the brain was immersion fixed for24 h in freshly prepared 4% paraformaldehyde in 100 mM KPO₄ (pH 7.2) forhistopathology. The hemi-brains were then incubated for 24 h in 10%, 20%and 30% sucrose sequentially for cyroprotection. Horizontal sections of25μ thickness were collected using a sliding microtome and stored at 4°C. in Dulbecco's phosphate-buffered saline with sodium azide (pH 7.2) toprevent microbial growth. A series of 8 equally spaced tissue sections600μ apart were randomly selected spanning the entire brain and stainedusing free-floating immunohistochemistry for total Aβ (rabbit polyclonalanti-pan Aβ; Biosource, Camarillo, Calif., 1:10,000) as previouslydescribed. Gordon et al., Exp. Neurol. 173:183-195 (2002); Wilcock etal., J. Neurosci. 24:6144-6151 (2004). A second series of tissuesections 600 μm apart were stained using 0.2% Congo red inNaCl-saturated 80% ethanol. Another set of sections were also mountedand stained for hemosiderin using 2% potassium ferrocyanide in 2%hydrochloric acid for 15 min, followed by a counterstain in a 1% neutralred solution for 10 min. Quantification of Congo red staining and Aβimmunohistochemistry was performed using the Image-Pro Plus (MediaCybernetics, Silver Spring, Md.) to analyze the percent area occupied bypositive stain. One region of the frontal cortex and three regions ofthe hippocampus were analyzed (to ensure that there was no regional biasin the hippocampal values). The initial analysis of Congo red wasperformed to give a total value. A second analysis was performed aftermanually editing out all of the parenchymal amyloid deposits to yield apercent area restricted to vascular Congo red staining. To estimate theparenchymal area of Congo red, the vascular amyloid values weresubtracted from the total percentage. For the hemosiderin stain thenumbers of Prussian blue-positive sites were counted on all sections andthe average number of sites per section calculated. Qualitativedifferences between animals were observed at the sections at a lowmagnification. Eight equally spaced sections were examined and thenumber of positive profiles was determined and averaged to a per-sectionvalue. To assess possible treatment-related differences, the values foreach treatment group were analyzed by one-way ANOVA followed by Fisher'sLSD means comparisons.

Measurement of Serum Level of Aβ Peptide Using ELISA.

Serum collected one day after the last dosing of antibodies was dilutedand incubated in 96-well microtiter plates (MaxiSorp; Nunc, Rosklide,Denmark), which were precoated with antibody 6E10 (anti-beta amyloidantibody that binds to Aβ₁₋₁₇; Signet, Dedham, Mass.) at 5 ug/ml in PBSbuffer, pH 7.4. The secondary antibody was biotinylated 4G8 (anti-betaamyloid antibody that binds to Aβ₁₇₋₂₄; Signet) at a 1:5000 dilution.Detection was done using a streptavidin-horseradish peroxidase conjugate(Amersham Biosciences), followed by TMB substrate (KPL, Gaithersburg,Md.). Aβ₁₋₄₀ (American Peptide) scaling from 6-400 pM were used forstandard curves.

B. Results

Reversal of Cognitive Deficits by Administration of DeglycosylatedAntibody.

The radial-arm water-maze task detects spatial learning and memorydeficits in transgenic mouse models. Gordon et al., Neurobiol. Aging.22:377-385 (2001); Morgan et al., Nature 408:982-985 (2000). Animaltreated with antibody 2H6, deglycosylated 2H6, or anti-AMN for 16 weekswere tested for spatial navigation learning in a two-day version of theradial-arm water maze. Nontransgenic normal mice (including 2 micetreated with 2H6 antibody and 4 mice treated with anti-AMN antibody;these two groups were combined since no behavior difference wasobserved) were also tested in the two-day version of the radial-armwater maze. As shown in FIG. 5, APP-transgenic mice treated with thecontrol antibody (anti-AMN) failed to learn platform location over twodays of testing and were significantly impaired compared to thenontransgenic mice as previously described. Wilcock et al., J.Neuroinflammation 1:24 (2004). However, APP-transgenic mice administeredthe anti-Aβ antibody 2H6 and deglycosylated 2H6 demonstrated asignificant reversal of the impairment observed in the control-treatedAPP-transgenic mice, ending day two with a mean performance near 1 errorper trial (FIG. 5). The control-treated APP-transgenic mice has a meanperformance near 3 errors per trial at the ending of day two (FIG. 5).The data shown in FIG. 5 indicate that the deglycosylated antibody worksas well as the intact antibody for reversal of cognitive deficits in theAPP-transgenic mice.

Reduction of Aβ Deposits without Increasing Microhemorrhage.

As shown Table 7 below, total Aβ immunostaining in the hippocampus wassignificantly reduced after 16 weeks immunotherapy with antibody 2H6(about 56% reduction, p=0.0001) and deglycosylated 2H6 (about 58%reduction, p<0.0001) as compared to the control antibody-treated group(anti-AMN). As shown in Table 8 below, total Aβ immunostaining in thefrontal cortex was significantly reduced after 16 weeks immunotherapywith antibody 2H6 (about 50% reduction, p<0.0001) and deglycosylated 2H6(about 51% reduction, p<0.0001) as compared to the controlantibody-treated group (anti-AMN).

TABLE 7 Total Aβ load for hippocampus after 16 weeks of antibodytreatment. Mean percent area occupied by positive immunohistochemicalstain for Aβ, and standard deviation and standard error of the mean forthe hippocampus are shown. Antibody Number of administered animalsanalyzed Mean Std. Dev. Std. Error Anti-AMN 6 27.127 4.602 1.879 2H6 412.011 5.057 2.529 Deglycosylated 2H6 5 11.344 4.765 2.131

TABLE 8 Total Aβ load for frontal cortex after 16 weeks of antibodytreatment. Mean percent area occupied by positive immunohistochemicalstain for Aβ, and standard deviation and standard error of the mean forthe frontal cortex are shown. Antibody Number of administered animalsanalyzed Mean Std. Dev. Std. Error Anti-AMN 6 47.060 4.667 1.905 2H6 423.708 6.355 3.178 Deglycosylated 2H6 5 22.834 1.970 0.881

As shown Table 9, total Congo-red staining in the hippocampus wassignificantly reduced after 16 weeks immunotherapy with antibody 2H6(about 77% reduction, p<0.0001) and deglycosylated 2H6 (about 53%reduction, p<0.0001) as compared to the control antibody-treated group(anti-AMN). As shown in Table 10, total Congo-red staining in thefrontal cortex was also significantly reduced after 16 weeksimmunotherapy with antibody 2H6 (about 79% reduction, p<0.0001) anddeglycosylated 2H6 (about 68% reduction, p<0.0001) as compared to thecontrol antibody-treated group (anti-AMN).

TABLE 9 Total Congo-red staining for hippocampus after 16 weeks ofantibody treatment. Mean percent area occupied by positive Congo-redstain for Aβ, and standard deviation and standard error of the mean forthe hippocampus are shown. Antibody Number of administered animalsanalyzed Mean Std. Dev. Std. Error Anti-AMN 6 1.210 0.081 0.033 2H6 40.281 0.021 0.010 Deglycosylated 2H6 5 0.573 0.101 0.045

TABLE 10 Total Congo-red staining for frontal cortex after 16 weeks ofantibody treatment. Mean percent area occupied by positive Congo-redstain for Aβ, and standard deviation and standard error of the mean forthe frontal cortex are shown. Antibody Number of administered animalsanalyzed Mean Std. Dev. Std. Error Anti-AMN 6 2.507 0.691 0.282 2H6 40.520 0.047 0.023 Deglycosylated 2H6 5 0.807 0.104 0.046

Parenchymal (Tables 11 and 12; and FIG. 6) and vascular (Tables 13 and14; FIG. 7) Congo-red staining were analyzed separately for both frontalcortex and hippocampus. As shown Table 11 and FIG. 6A, parenchymalCongo-red staining in the frontal cortex was significantly reduced after16 weeks immunotherapy with antibody 2H6 (about 98% reduction, p<0.0001)and deglycosylated 2H6 (about 77% reduction, p<0.0001) as compared tothe control antibody-treated group (anti-AMN). As shown in Table 12 andFIG. 6B, parenchymal Congo-red staining in the hippocampus was alsosignificantly reduced after 16 weeks immunotherapy with antibody 2H6(about 96% reduction, p<0.0001) and deglycosylated 2H6 (about 63%reduction, p<0.0001) as compared to the control antibody-treated group(anti-AMN). The deglycosylated 2H6 was less effective than the intact2H6 antibody in reducing Congo-red load in the frontal cortex andhippocampus.

TABLE 11 Parenchymal Congo-red staining for frontal cortex after 16weeks of antibody treatment. Mean percent area occupied by positiveCongo-red stain for Aβ, and standard deviation and standard error of themean for the frontal cortex are shown. Antibody Number of administeredanimals analyzed Mean Std. Dev. Std. Error Anti-AMN 6 2.360 0.676 0.2762H6 4 0.059 0.047 0.024 Deglycosylated 2H6 5 0.537 0.144 0.064

TABLE 12 Parenchymal Congo-red staining for hippocampus after 16 weeksof antibody treatment. Mean percent area occupied by positive Congo-redstain for Aβ, and standard deviation and standard error of the mean forthe hippocampus are shown. Antibody Number of administered animalsanalyzed Mean Std. Dev. Std. Error Anti-AMN 6 1.117 0.104 0.043 2H6 40.040 0.029 0.015 Deglycosylated 2H6 5 0.416 0.078 0.035

As shown Table 13 and FIG. 7A, vascular Congo-red staining in thehippocampus was significantly increased after 16 weeks immunotherapywith antibody 2H6 (about 2.7 fold, p<0.0001) and deglycosylated 2H6(about 1.7 fold, p=0.0185) as compared to the control antibody-treatedgroup (anti-AMN). As shown in Table 14 and FIG. 7B, vascular Congo-redstaining in the frontal cortex was also significantly increased after 16weeks immunotherapy with antibody 2H6 (about 3.5 fold, p<0.0001) anddeglycosylated 2H6 (about 1.8 fold, p=0.0048) as compared to the controlantibody-treated group (anti-AMN). The increase of vascular Congo-redstaining in the deglycosylated 2H6 treated group was significantly lessthan in the intact 2H6 antibody treated group for both hippocampus(p=0.0025) and frontal cortex (p<0.0001).

TABLE 13 Vascular Congo-red staining for hippocampus after 16 weeks ofantibody treatment. Mean percent area occupied by positive Congo-redstain for Aβ, and standard deviation and standard error of the mean forthe hippocampus are shown. Antibody Number of administered animalsanalyzed Mean Std. Dev. Std. Error Anti-AMN 6 0.093 0.036 0.015 2H6 40.253 0.053 0.027 Deglycosylated 2H6 5 0.157 0.030 0.013

TABLE 14 Vascular Congo-red staining for frontal cortex after 16 weeksof antibody treatment. Mean percent area occupied by positive Congo-redstain for Aβ, and standard deviation and standard error of the mean forthe frontal cortex are shown. Antibody Number of administered animalsanalyzed Mean Std. Dev. Std. Error Anti-AMN 6 0.147 0.055 0.023 2H6 40.511 0.084 0.042 Deglycosylated 2H6 5 0.269 0.043 0.019

Prussian blue histological stain was used to label hemosiderin, a fericoxide material produced in the breakdown of hemoglobin. Extravenousblood in the brain leads to microglial phagocytosis of the erythrocytesand breakdown of the hemoglobin within them. Thus, ferricoxide-containing microglia are thus markers of past hemorrhage. Thenumber Prussian blue-positive profiles in antibody treated animals werecounted. As shown in Table 15 and FIG. 8, treatment with antibody 2H6significantly (p<0.0001) increased Prussian blue staining for about 5.5fold as compared to the control antibody treated group (anti-AMN).Treatment with deglycosylated 2H6 antibody only increased Prussian bluestaining for about 1.8 fold (p=0.0364) as compared to the controlantibody treated group.

TABLE 15 Prussian blue staining for the entire section after 16 weeks ofantibody treatment. Mean percent area occupied by positive Prussian bluestain, and standard deviation and standard error of the mean for theentire section are shown. Antibody Number of administered animalsanalyzed Mean Std. Dev. Std. Error Anti-AMN 7 0.589 0.295 0.112 2H6 43.250 0.445 0.222 Deglycosylated 2H6 5 1.050 0.143 0.064

Serum Level of Aβ after Administration of Deglycosylated 2H6 Antibody.

As shown in FIG. 9, administration of both antibody 2H6 anddeglycosylated 2H6 to the APP Tg2576 mice significantly increased serumlevel of beta-amyloid peptide. However, no significant increase of serumlevel of beta-amyloid peptide was observed in the APP Tg2576 mice afteradministration of anti-AMN antibody or in the wild type mice afteradministration of anti-AMN antibody or antibody 2H6. This indicates thatthe increase of serum level of beta-amyloid peptide may be used toassist diagnosis of AD and monitor the response to AD therapy, such asimmunotherapy.

C. Conclusion

The above data demonstrate that 1) the deglycosylated antibody 2H6 is aseffective as the intact antibody 2H6 in reversing learning and memorydeficits in APP Tg2576 mice; 2) the deglycosylated antibody 2H6 is alittle less effective as the intact antibody 2H6 in depleting Aβdeposits in hippocampus and frontal cortex as measured by Congo-redstaining, but is as effective in depleting Aβ load in hippocampus andfrontal cortex as measured by Aβ immunostaining; 3) increase of Aβdeposit (as measured by Congo-red staining) in hippocampous and frontalcortex vasculature by the deglycosylated antibody 2H6 is much less thanthe intact 2H6 antibody; and 4) microhemorrhage as measured by Prussianblue staining in APP Tg2576 mice treated with the deglycosylated 2H6antibody was much lower than mice treated with the intact 2H6 antibody.These data suggest that deglycosylated antibody is as effective forimproving indications of Alzheimer's disease with lower risk ofmicrohemorrhage in APP Tg2576 mice.

Example 5 Binding Affinity of Various Antibody Fc Regions to Murine andHuman Fcγ Receptors and Complement

Binding affinity of the antibody Fc regions to Fcγ receptors orcomplement were measured using BIAcore as described above. Briefly,purified human or murine Fcγ receptors (from R&D Systems) and human Clq(from Quidel) were immobilized on BIAcore CM5 chip by amine chemistry.Serial dilutions of monoclonal antibodies (ranging from 2 nM to themaximum concentration as indicated in Tables 16 and 17) were injected.HBS-EP (0.01M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% SurfactantP20) as running and sample buffer. Binding data were analyzed using 1:1langmuir interaction model for high affinity interactions, or steadystate affinity model for low affinity interactions.

Table 16 below shows the binding affinity of the anti-β-amyloidantibodies as measured by K_(D) (nM) to murine FcγRI, FcγRIIb, FcγRIII,and human Clq (hClq). Deglycosylated antibodies have a constant regionwith removed N-glycosylation. 9TL(hIgG1) and 9TL(hIgG2Aa) have the samevariable region (shown in SEQ ID NO:1 and SEQ ID NO:2), but differentconstant region. 9TL(hIgG1) has a human IgG1 constant region; and9TL(hIgG2Aa) has a human IgG2a with mutations of A330P331 to S330S331(Kabat amino acid numbering with reference to the wildtype IgG2asequence). As shown in Table 16, deglycosylated 2H6, 2294, and 2286 hadreduced affinity to all murine Fcγ receptors tested as compared to eachcorresponding antibody without removed N-glycosylation. Deglycosylated2H6 also had reduced affinity to human complement as compared to 2H6.9TL(hIgG1) had no significant binding to mFcγRIIb or mFcγRIII; and9TL(hIgG2Aa) had no significant binding to any of murine FcγRI, FcγRIIb,FcγRIII, or hClq.

Table 17 below shows the binding affinity of the anti-β-amyloidantibodies as measured by K_(D) (nM) to human FcγRI, FcγRIIb/c,FcγRIIIb, and hClq. As shown in Table 17, 9TL(hIgG2Aa) had nosignificant binding to human FcγRI or hClq; and significant loweraffinity to human FcγRIIb/c and FcγRIIIb as compared to the affinity ofthe antibody with human IgG1 constant region to these molecules.

TABLE 16 Binding affinity of antibodies to murine Fcγ receptors andhuman complement as measured by K_(D) (nM) Maximum antibodyconcentration tested for binding to Fcγ receptors Antibody FcγRI FcγRIIbFcγRIII hC1q Isotype (nM) 2H6 1,800 76,000 133,000  5,000 Murine IgG2b49,000 Deglycosylated 5,600 NB NB 30,000 Deglycosylated 17,000 2H6murine IgG2b 2294 1,200 13,000 19,000 Murine IgG2b 18,000 Deglycosylated8,600 NB NB Deglycosylated 22,000 2294 murine IgG2b 2286 93  5,00010,000 Murine IgG1 12,000 Deglycosylated 2,700 NB NB Deglycosylated9,300 2286 murine IgG1 9TL(hIgG1) 800 NB NB 34 Human IgG1 30,0009TL(hIgG2AΔa) NB NB NB NB Human IgG2Δa 30,000 NB: no significant bindingwhen antibody was used at the maximum concentration tested. Maximumantibody concentration tested for binding to hC1q was 30,000 nM.

TABLE 17 Binding affinity of antibodies to human Fcγ receptors and humancomplement as measured by K_(D) (nM) Antibody FcγRI FcγRIIb/c FcγRIIIbhC1q 9TL(hIgG1) 2.2 7,000 33,000 34 9TL(hIgG2Δa) NB 61,000 >100,000 NBNB: no significant binding when antibody is used at 30 μM concentration.Maximum antibody concentration tested for binding was 30,000 nM.

Example 6 Effect of Deglycosylated 2H6 Antibody on MicroglialActivation, Fcγ Receptor Binding, and Amyloid Clearance AfterIntracranial Administration

Surgical Procedure and Intracranial Administration of Antibodies

Tg2576 transgenic mice aged 19.5 months were assigned to one of thethree groups, all groups received intracranial injections into thefrontal cortex and hippocampus. The first group received anti-Aβ-antibody 2H6 at a concentration of 2 μg/2 μl in each region. The secondgroup received deglycosylated 2H6 antibody at 2 μg/2 μl in each region.The third group received IgG directed against drosophila amnesiacprotein as a control for nonspecific aspects of intact IgG injection.All mice survived for 72 hours after surgery.

On the day of surgery the mice (Tg2576 transgenic mice) were weighed,anesthetized with isoflurane and placed in a stereotaxic apparatus(51603 dual manipulator lab standard, Stoelting, Wood Dale, Ill.). Amidsagittal incision was made to expose the cranium and two burr holeswere drilled using a dental drill over the right frontal cortex andhippocampus to the following coordinates: Cortex: AP +1.5 mm, L −2.0 mm,hippocampus: AP −2.7 mm, L −2.5 mm, all taken from bregma. A 26 gaugeneedle attached to a 10 μl Hamilton (Reno, Nev.) syringe was lowered 3mm ventral to bregma and a 2 μl injection was made over a 2 minuteperiod. The incision was cleaned with saline and closed with surgicalstaples.

Tissue preparations. On the day of sacrifice mice were weighed,overdosed with 100 mg/kg pentobarbital (Nembutal sodium solution, Abbottlaboratories, North Chicago Ill.) and intracardially perfused with 25 ml0.9% sodium chloride followed by 50 ml freshly prepared 4%paraformaldehyde (pH=7.4). Brains were rapidly removed and immersionfixed for 24 hours in freshly prepared 4% paraformaldehyde. The brainswere then incubated for 24 hours in 10, 20 and 30% sucrose sequentiallyto cyroprotect them. Horizontal sections of 25 μm thickness were thencollected using a sliding microtome and stored at 4° C. in DPBS bufferwith sodium azide to prevent microbial growth.

Six to eight sections approximately 100 μm apart were selected spanningthe injection site and stained using free-floating immunohistochemistrymethods for total Aβ (rabbit antiserum reacting with Aβ₁₋₄₀ and Aβ₁₋₄₂;dilution used 1:10,000), anti-CD45 antibody (Cat. No. MCA1031G, Serotec,Raleigh N.C.; dilution used 1:5000), and anti-Fcγ receptor (CD16/CD32)antibody (Cat. No. 553141 from BD Biosciences; used as 1:1,000dilution). Biotinylated goat anti-rabbit at 1:3,000 dilution was used asthe secondary antibody for anti-Aβ antibody staining Biotinylated goatanti-rat at 1:3,000 dilution was used as the secondary antibody foranti-CD45 and Fcγ receptor antibody staining. For immunostaining, somesections were omitted from the primary antibody to assess non-specificimmunohistochemical reactions. Adjacent sections were mounted on slidesand stained using 4% thioflavine-S (Sigma-Aldrich, St Louis Mo.) for 10minutes. It should be noted that there were a limited number of sectionsthat include the injection volume.

Data analysis. The immunohistochemical reaction product on all stainedsections was measured using a videometric V150 image analysis system(Oncor, San Diego, Calif.) in the injected area of cortex andhippocampus and corresponding regions on the contralateral side of thebrain. Data were presented as the ratio of injected side to non-injectedside for Aβ staining, thioflavine-S staining, CD45 staining, and Fcγreceptor staining Normalizing each injection site to the correspondingcontralateral site diminishes the influence of interanimal variabilityand permits reliable measurements of drug effects with a smaller numberof mice. To assess possible treatment-related differences, the ratiovalues for each treatment group were analyzed by ANOVA using StatViewsoftware version 5.0.1 (SAS Institute Inc., NC) followed by Fischer'sLSD means comparisons.

Results

As shown in FIG. 10, CD45 staining in the frontal cortex and thehippocampus was about the same after intracranial administration ofdeglycosylated 2H6 antibody as the control antibody. In contrast, CD45staining in the frontal cortex was significantly higher (p<0.01) and wasgenerally higher in the hippocampus after intracranial administration of2H6 antibody than the control antibody. This indicates that, unlikeantibody 2H6, administration of deglycosylated 2H6 did not activatemicroglia in the frontal cortex and the hippocampus 72 hours after theadministration.

As shown in FIG. 11, FcγII and FcγIII receptor staining in the frontalcortex and the hippocampus was about the same after intracranialadministration of deglycosylated 2H6 antibody as the control antibody.In contrast, Fcγ receptor staining in the frontal cortex and thehippocampus was significantly higher (p<0.01) after intracranialadministration of 2H6 antibody than the control antibody. This indicatesthat, unlike antibody 2H6, administration of deglycosylated 2H6 did notactivate microglia in the frontal cortex and the hippocampus 72 hoursafter the administration.

As shown in FIG. 12, Aβ staining was lower in frontal cortex andhippocampus 72 hours after intracranial administration of 2H6 antibodyor deglycosylated 2H6 antibody as compared to the control antibody. Asshown in FIG. 13, thioflavin-S stained compact plaque was also lower infrontal cortex and hippocampus 72 hours after intracranialadministration of 2H6 antibody or deglycosylated 2H6 antibody ascompared to the control antibody.

These data indicate that deglycosylated 2H6 antibody was able to reduceAβ and compact plaques in frontal cortex and hippocampus withoutinducing microglia activation and inflammatory response.

Example 7 Characterization of Epitope on Aβ Peptide that Antibody 2294Binds

Antibody 2294 is a murine antibody raised by immunizing a mouse withAβ₁₋₄₀. This antibody is described in US 2004/0146512 and WO 04/032868.

Binding affinity for antibody 2294 to Aβ₁₋₄₀, Aβ₁₋₄₂, or Aβ₂₂₋₃₇ wasmeasured using Biacore as described above. Table 18 below shows theaffinity of antibody 2294 Fab fragment to various Aβ peptides.

TABLE 18 Binding affinity of antibody 2294 Fab fragment K_(D) k_(on)(1/Ms) K_(off) (1/s) (nM) Biotinylated Aβ₁₋₄₀ immobilized on 6.6 × 10⁴3.95 × 10⁻⁴ 6 streptavidin chip, 2294 Fab flowed onto it BiotinylatedAβ₁₋₄₂ immobilized on 1.1 × 10⁴ 4.87 × 10⁻³ 400 streptavidin chip, 2294Fab flowed onto it Biotinylated Aβ₂₂₋₃₇ immobilized on   5 × 10³ 0.04910,000 streptavidin chip, 2294 Fab flowed onto it

Epitope mapping of antibody 2294 was performed by ELISA assay.Biotinylated 15-mer or 10-mer of various Aβ peptides (these peptideshave glycine added to the C-terminal end) were immobilized onstreptavidin coated plates. NUNC maxisorp plates were coated with 6ug/ml of streptavidin (Pierce, 21122) in PBS pH 7.4 for more than 1 h at4° C. Plates were blocked with 1% BSA in PBS buffer pH 7.4. Afterwashing, biotinylated Aβ peptides in PBS pH 7.4 were incubated 1 hour atroom temperature. Antibody 2294 (from 2.5 ug/ml to 10 ug/ml) wasincubated with the immobilized Aβ peptides for 1 h at room temperature.After washing, plates were incubated with secondary antibody, a HRPconjugated goat anti-human kappa chain antibody (MP Biomedicals, 55233)at 1:5000 dilution. After washing, bound secondary antibody was measuredby adding TMB substrate (KPL, 50-76-02, 50-65-02). HRP reaction wasstopped by adding 1M phosphoric acid and absorbance at 450 nm wasmeasured. As shown in FIG. 14, antibody 2294 binds to Aβ peptides withamino acids 20-34, 21-35, 22-36, 23-37, 24-38, 25-39, 26-40, and 25-34with a glycine at the C-terminus; but does not bind to Aβ peptides withamino acids 19-33, 27-41, 24-33, and 27-35 having a glycine at theC-terminus of these peptides. This suggests that the epitope of antibody2294 binds includes amino acids from 26 to 34.

To further determine the epitope on Aβ peptide that is recognized byantibody 2294, ELISA binding analysis was used. Various Aβ peptides(Global Peptide Services, CO) was immobilized on a ELISA plate. Thebinding of 2294 full antibody (at 20 nM) to the immobilized Aβ wasdetermined by ELISA as described above. Antibody 2294 binds to Aβpeptides 17-40, 17-42, 28-40, 1-38, 1-40, 1-42, and 1-43. Antibody 2294did not bind to Aβ peptide 1-16, 1-28, 28-42, 22-35, and 33-40. Thus,antibody 2294 binds to the C-terminus of various truncated Aβ peptide,for example, 1-38, 1-40, 1-42, and 1-43.

Table 19 below shows binding comparison of 2294 to Aβ₁₋₄₀ to other Aβpeptide as measured by Biacore assay. Antibody 2294 (full antibody) hasthe strongest binding to Aβ₁₋₄₀ as compared to other peptides, withsignificantly lower binding to truncated Aβ₁₋₄₀ (such as 1-36, 1-37,1-38, and 1-39), Aβ₁₋₄₂ and Aβ₁₋₄₃. This indicates that the side chainor backbone of amino acid 40 (Valine) of Aβ is involved in binding of2294 to Aβ₁₋₄₀; and binding is significantly reduced in absence of thisamino acid.

TABLE 19 Aβ peptide fragment Binding 1-28 − 1-43 − 22-35  − 1-36 +1-37 + 1-38 ++ 1-39 ++ 17-42  +++ 1-42 +++ 17-40  ++++ 1-40 ++++ “−”indicates no binding; “+” indicates very low binding; “++” indicatesmedium binding; “+++” indicates strong binding; and “++++” indicatesvery strong binding.

Based on data shown above, the epitope that antibody 2294 binds seems toinclude amino acids 26-34 and 40. Antibody 2294 binds to an epitope verysimilar to antibody 6G described in U.S. provisional application Ser.No. 60/676,093 (the amino acid and nucleic acid sequences of thisantibody is shown in SEQ ID NOS:36-39; vectors encoding 6G are depositedat American Type Culture Collection on Jun. 15, 2005, with accessionnumbers PTA-6786 and PTA-6787). The epitope that antibody 6G bindsincludes amino acids from 25 to 34, and 40. The epitope comparison ofantibody 2294 and 6G are shown in FIG. 14.

The binding affinity of antibody 6G Fab fragment to Aβ₁₋₄₀, Aβ₁₋₄₂, orAβ₂₂₋₃₇ was measured using Biacore. Biotinylated Aβ₁₋₄₀, Aβ₁₋₄₂, orAβ₂₂₋₃₇ was immobilized on a streptavidin chip, and 6G Fab was flowedonto it. Antibody 6G Fab fragment binds to Aβ₁₋₄₀ with k_(on) (1/Ms) of3.0×10⁵, k_(off) (1/s) of 7.0×10⁻⁴, and K_(D) of 2 nM. Antibody 6G Fabfragment binds to Aβ₁₋₄₂ with k_(on), (1/Ms) of 1.8×10⁴, k_(off) (Vs) of1.6×10⁻³, and K_(D) of 80 nM. Antibody 6G Fab fragment binds to Aβ₂₂₋₃₇with k_(on) (1/Ms) of 3.6×10⁵, k_(off) (1/s) of 3.9×10⁻³, and K_(D) of11 nM. Antibody 6G has significant higher affinity to Aβ₁₋₄₂, andAβ₂₂₋₃₇ than antibody 2294. Data indicate that binding of antibody 6G isless dependent on amino acid 40 than antibody 2294.

Antibody competition experiments between 2294, 6G, 2H6, and 2289 usingBiacore assay were performed as described in Example 3. Competitionexperiments were performed using Biacore assay. Antibody 2294, 6G, 2H6,and 2289 were immobilized on different channels of a CM5 chip. CM5 chipchannels were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antibody 2294, 6G, 2H6, and 2289 were each diluted into 10 mM sodiumacetate pH 4.0 and injected over an activated chip at a concentration of0.005 mg/mL. Each channel was blocked with ethanolamine. Aβ₁₋₄₀ peptide(150 uM) was flowed onto the chip for 2 min. Then antibody 2294 (to betested for competition of binding) at 0.6 uM was flowed onto the chipfor 1 min. HBS-EP buffer (0.01M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA,0.005% Surfactant P20) was used as running buffer for all the BIAcoreassays. After measuring binding of Aβ₁₋₄₀, all channels of the chip wereregenerated by washing twice with a mixture of Pierce elution buffer(Product No. 21004, Pierce Biotechnology, Rockford, Ill.) and 4 M NaCl(2:1) for 6 sec. Competition binding was then performed for antibody 6G,2H6, and then antibody 2289. Competition between 2294 and 6G and between2294 and 2H6 for binding to Aβ₁₋₄₀ was observed, but no competition wasobserved between 2294 and 2289 or between 6G and 2289. Observations ofcompetition between the antibody immobilized and the same antibodyflowed onto the chip served as the positive control. Data indicate thatantibody 2294 competes with 2H6 and 6G for binding to Aβ₁₋₄₀.

Example 8 Effect of Antibody 2294 and Deglycosylated Antibody 2294 inReducing Aβ Deposit and Cognition In Animal Model of Alzheimer's Disease

Deglycosylated antibody 2294 was prepared as by incubating purifiedantibody 2294 at 37° C. for 7 days with peptide-N-glycosidase F(Prozyme, 0.05 Upper mg of antibody) in 20 mM Tris-HCl pH 8.0.Completeness of deglycosylation was verified by MALDI-TOF-MS and proteingel electrophoresis. Deglycosylated antibodies were purified by ProteinA chromatography and endotoxin was removed by Q-Sepharose. The bindingaffinity to Aβ₁₋₄₀ of the deglycosylated 2294 was tested using Biacoreassay described above, and the binding affinity of the deglycosylated2294 to Aβ₁₋₄₀ was found to be identical to the intact antibody 2294.

Antibody 2294 and deglycosylated 2294 were tested in transgenic mice APPTg2576 for effect on reversal of cognitive deficits, histologicalsymptoms, and microhemorrhage as described in Example 4. For the sixteenweeks treatment study, transgenic mice (aged 20 months) were assigned towere assigned to one of the four groups. The first group received weeklyintraperitoneal anti-Aβ antibody 2294 injections for a period of 16weeks (n=4). The second group received weekly intraperitonealdeglycosylated anti-Aβ antibody 2294 injections for a period of 16 weeks(n=5). The third group received weekly intraperitoneal anti-AMN antibody(2906; mouse-monoclonal anti-Drosophila amnesiac protein IgG1)injections for a period of 16 weeks (n=6). Non-transgenic littermateswere treated for 16 weeks with either anti-AMN antibody (n=4) or 2294(n=2).

Histological and behavioral analysis are performed as described inExample 4.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application. Allpublications, patents and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent or patent applicationwere specifically and individually indicated to be so incorporated byreference.

Deposit of Biological Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, USA (ATCC):

ATCC Material Antibody No. Accession No. Date of Deposit pDb.9TL.hFc2a9TL heavy chain PTA-6124 Jul. 20, 2004 pEb.9TL.hK 9TL light chainPTA-6125 Jul. 20, 2004 pDb.6G.hFc2a 6G heavy chain PTA-6786 Jun. 15,2005 pEb.6G.hK 6G light chain PTA-6787 Jun. 15, 2005

Vector pEb.9TL.hK is a polynucleotide encoding the 9TL light chainvariable region and the light chain kappa constant region; and vectorpDb.9TL.hFc2a is a polynucleotide encoding the 9TL heavy chain variableregion and the heavy chain IgG2a constant region containing thefollowing mutations: A330P331 to S330S331 (amino acid numbering withreference to the wildtype IgG2a sequence; see Eur. J. Immunol. (1999)29:2613-2624).

Vector pEb.6G.hK is a polynucleotide encoding the 6G light chainvariable region and the light chain kappa constant region; and vectorpDb.6G.hFc2a is a polynucleotide encoding the 6G heavy chain variableregion and the heavy chain IgG2a constant region containing thefollowing mutations: A330P331 to S330S331 (amino acid numbering withreference to the wildtype IgG2a sequence; see Eur. J. Immunol. (1999)29:2613-2624).

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Rinat Neuroscience Corp. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC Section 122 and the Commissioner's rulespursuant thereto (including 37 CFR Section 1.14 with particularreference to 8860G 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

Antibody sequences 9TL heavy chain variable region amino acid sequence(SEQ ID NO: 1) QVQLVQSGAEVKKPGASVKVSCKASGYYTEAYYIHWVRQAPGQGLEWMGRIDPATGNTKYAPRLQDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASLYSLPVYWGQGTT VTVSS9TL light chain variable region amino acid sequence (SEQ ID NO: 2)DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDAKTYLNWFQQRPGQSPRRLIYQISRLDPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGTHYPVLFGQGTRLEIKRT9TL CDR H1 (extended CDR) (SEQ ID NO: 3) GYYTEAYYIH9TL CDR H2 (extended CDR) (SEQ ID NO: 4) RIDPATGNTKYAPRLQD9TL CDR H3 (extended CDR) (SEQ ID NO: 5) LYSLPVY9TL CDR Ll (extended CDR) (SEQ ID NO: 6) KSSQSLLYSDAKTYLN9TL CDR L2 (extended CDR) (SEQ ID NO: 7) QISRLDP9TL CDR L3 (extended CDR) (SEQ ID NO: 8) LQGTHYPVL9TL heavy chain variable region nucleotide sequence (SEQ ID NO: 9)CAGGTGCAGCTGGTGCAGTCTGGTGCTGAGGTGAAGAAGCCTGGCGCTTCCGTGAAGGTTTCCTGCAAAGCATCTGGTTACTATACGGAGGCTTACTATATCCACTGGGTGCGCCAAGCCCCTGGTCAAGGCCTGGAGTGGATGGGCAGGATTGATCCTGCGACTGGTAATACTAAATATGCCCCGAGGTTACAGGACCGGGTGACCATGACTCGCGATACCTCCACCAGCACTGTCTACATGGAACTGAGCTCTCTGCGCTCTGAGGACACTGCTGTGTATTACTGTGCCTCCCTTTATAGTCTCCCTGTCTACTGGGGCCAGGGTACCACTGTTACCGTGTCC TCT9TL light chain variable region nucleotide sequence (SEQ ID NO: 10)GATGTTGTGATGACCCAGTCCCCACTGTCTTTGCCAGTTACCCTGGGACAACCAGCCTCCATATCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTGATGCCAAGACATATTTGAATTGGTTCCAACAGAGGCCTGGCCAGTCTCCACGCCGCCTAATCTATCAGATTTCCCGGCTGGACCCTGGCGTGCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTTAAAATCAGCAGAGTGGAGGCTGAAGATGTGGGAGTTTATTACTGCTTACAAGGTACACATTATCCGGTGCTCTTCGGTCAAGGGACCCGCCTGGAGATCAAACGC ACT9TL heavy chain full antibody amino acid sequence(including modified IgG2a as described herein) (SEQ ID NO: 11)QVQLVQSGAEVKKPGASVKVSCKASGYYTEAYYIHWVRQAPGQGLEWMGRIDPATGNTKYAPRLQDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASLYSLPVYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK9TL light chain full antibody amino acid sequence (SEQ ID NO: 12)DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDAKTYLNWFQQRPGQSPRRLIYQISRLDPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGTHYPVLFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC9TL heavy chain full antibody nucleotide sequence(including modified IgG2a as described herein) (SEQ ID NO: 13)CAGGTGCAGCTGGTGCAGTCTGGTGCTGAGGTGAAGAAGCCTGGCGCTTCCGTGAAGGTTTCCTGCAAAGCATCTGGTTACTATACGGAGGCTTACTATATCCACTGGGTGCGCCAAGCCCCTGGTCAAGGCCTGGAGTGGATGGGCAGGATTGATCCTGCGACTGGTAATACTAAATATGCCCCGAGGTTACAGGACCGGGTGACCATGACTCGCGATACCTCCACCAGCACTGTCTACATGGAACTGAGCTCTCTGCGCTCTGAGGACACTGCTGTGTATTACTGTGCCTCCCTTTATAGTCTCCCTGTCTACTGGGGCCAGGGTACCACTGTTACCGTGTCCTCTGCCTCCACCAAGGGCCCATCTGTCTTCCCACTGGCCCCATGCTCCCGCAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCAGAACCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTGCAGTCCTCAGGTCTCTACTCCCTCAGCAGCGTGGTGACCGTGCCATCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCAAGCAACACCAAGGTCGACAAGACCGTGGAGAGAAAGTGTTGTGTGGAGTGTCCACCTTGTCCAGCCCCTCCAGTGGCCGGACCATCCGTGTTCCTGTTCCCTCCAAAGCCAAAGGACACCCTGATGATCTCCAGAACCCCAGAGGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGCAGTTCAACTGGTATGTGGACGGAGTGGAGGTGCACAACGCCAAGACCAAGCCAAGAGAGGAGCAGTTCAACTCCACCTTCAGAGTGGTGAGCGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGAAAGGAGTATAAGTGTAAGGTGTCCAACAAGGGACTGCCATCCAGCATCGAGAAGACCATCTCCAAGACCAAGGGACAGCCAAGAGAGCCACAGGTGTATACCCTGCCCCCATCCAGAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGATTCTATCCATCCGACATCGCCGTGGAGTGGGAGTCCAACGGACAGCCAGAGAACAACTATAAGACCACCCCTCCAATGCTGGACTCCGACGGATCCTTCTTCCTGTATTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGAAACGTGTTCTCTTGTTCCGTGATGCACGAGGCCCTGCACAACCACTATACCCAGAAGAGCCTGTCCCTGTCTCCAGGAAAGTAATTCTAGA9TL light chain full antibody nucleotide sequence (SEQ ID NO: 14)GATGTTGTGATGACCCAGTCCCCACTGTCTTTGCCAGTTACCCTGGGACAACCAGCCTCCATATCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTGATGCCAAGACATATTTGAATTGGTTCCAACAGAGGCCTGGCCAGTCTCCACGCCGCCTAATCTATCAGATTTCCCGGCTGGACCCTGGCGTGCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTTAAAATCAGCAGAGTGGAGGCTGAAGATGTGGGAGTTTATTACTGCTTACAAGGTACACATTATCCGGTGCTCTTCGGTCAAGGGACCCGCCTGGAGATCAAACGCACTGTGGCTGCACCATCTGTCTTCATCTTCCCTCCATCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCACGCGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACCCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCTCCAGTCACAAAGAGCTTCAACCGCGGTGAGTGCTAATTCTAG6G heavy chain variable region amino acid sequence (SEQ ID NO: 26)Q V Q L V Q S G A E V K K P G A S V K V S C K A S G Y T F T T Y A I H W V R QA P G Q G L E W M G F T S P Y S G V S N Y N Q K F K G R V T M T R D T S T S TV Y M E L S S L R S E D T A V Y Y C A R F D N Y D R G Y V R D Y W G Q G T L VT V S 6G light chain variable region amino acid sequence (SEQ ID NO: 27)D I V M T Q S P D S L A V S L G E R A T I N C R A S E S V D N D R I S F L N WY Q Q K P G Q P P K L L I Y A A T K Q G T G V P D R F S G S G S G T D F T L TI S S L Q A E D V A V Y Y C Q Q S K E F P W S F G G G T K V E I K R T V6G CDR H1 (extended CDR) (SEQ ID NO: 28) G Y T F T T Y A I H6G CDR H2 (extended CDR) (SEQ ID NO: 29)F T S P Y S G V S N Y N Q K F K G 6G CDR H3 (extended CDR)(SEQ ID NO: 30) F D N Y D R G Y V R D Y 6G CDR L1 (extended CDR)(SEQ ID NO: 31) R A S E S V D N D R I S F L N 6G CDR L2 (extended CDR)(SEQ ID NO: 32) A A T K Q G T 6G CDR L3 (extended CDR) (SEQ ID NO: 33)Q Q S K E F P W S 6G heavy chain variable region nucleotide sequence(SEQ ID NO: 34) CAGGTGCAACTGGTGCAATCCGGTGCCGAGGTGAAAAAGCCAGGCGCCTCCGTGAAAGTGTCCTGCAAAGCCTCCGGTTACACCTTTACCACCTATGCCATCCATTGGGTGCGCCAGGCCCCAGGCCAGGGTCTGGAGTGGATGGGCTTTACTTCCCCCTACTCCGGGGTGTCGAATTACAATCAGAAGTTCAAAGGCCGCGTCACCATGACCCGCGACACCTCCACCTCCACAGTGTATATGGAGCTGTCCTCTCTGCGCTCCGAAGACACCGCCGTGTATTACTGTGCCCGCTTCGACAATTACGATCGCGGCTATGTGCGTGACTATTGGGGCCAGGGCACCCTGGTCACCGTCTCC 6G light chain variable region nucleotide sequence(SEQ ID NO: 35)GACATCGTGATGACCCAGTCCCCAGACTCCCTGGCCGTGTCCCTGGGCGAGCGCGCCACCATCAACTGCCGCGCCAGCGAATCCGTGGATAACGATCGTATTTCCTTTCTGAACTGGTACCAGCAGAAACCAGGCCAGCCTCCTAAGCTGCTCATTTACGCCGCCACCAAACAGGGTACCGGCGTGCCTGACCGCTTCTCCGGCAGCGGTTCCGGCACCGATTTCACTCTGACCATCTCCTCCCTGCAGGCCGAAGATGTGGCAGTGTATTACTGTCAGCAGTCCAAAGAGTTTCCCTGGTCCTTTGGCGGTGGCACCAAGGTGGAGATCAAACGCACTG TG6G heavy chain full antibody amino acid sequence(including modified IgG2a as described herein) (SEQ ID NO: 36)QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYAIHWVRQAPGQGLEWMGFTSPYSGVSNYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARFDNYDRGYVRDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK6G light chain full antibody amino acid sequence (SEQ ID NO: 37)DIVMTQSPDSLAVSLGERATINCRASESVDNDRISFLNWYQQKPGQPPKLLIYAATKQGTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEFPWSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC6G heavy chain full antibody nucleotide sequence(including modified IgG2a as described herein) (SEQ ID NO: 38)CAGGTGCAACTGGTGCAATCCGGTGCCGAGGTGAAAAAGCCAGGCGCCTCCGTGAAAGTGTCCTGCAAAGCCTCCGGTTACACCTTTACCACCTATGCCATCCATTGGGTGCGCCAGGCCCCAGGCCAGGGTCTGGAGTGGATGGGCTTTACTTCCCCCTACTCCGGGGTGTCGAATTACAATCAGAAGTTCAAAGGCCGCGTCACCATGACCCGCGACACCTCCACCTCCACAGTGTATATGGAGCTGTCCTCTCTGCGCTCCGAAGACACCGCCGTGTATTACTGTGCCCGCTTCGACAATTACGATCGCGGCTATGTGCGTGACTATTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCTGTCTTCCCACTGGCCCCATGCTCCCGCAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCAGAACCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTGCAGTCCTCAGGTCTCTACTCCCTCAGCAGCGTGGTGACCGTGCCATCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCAAGCAACACCAAGGTCGACAAGACCGTGGAGAGAAAGTGTTGTGTGGAGTGTCCACCTTGTCCAGCCCCTCCAGTGGCCGGACCATCCGTGTTCCTGTTCCCTCCAAAGCCAAAGGACACCCTGATGATCTCCAGAACCCCAGAGGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGCAGTTCAACTGGTATGTGGACGGAGTGGAGGTGCACAACGCCAAGACCAAGCCAAGAGAGGAGCAGTTCAACTCCACCTTCAGAGTGGTGAGCGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGAAAGGAGTATAAGTGTAAGGTGTCCAACAAGGGACTGCCATCCAGCATCGAGAAGACCATCTCCAAGACCAAGGGACAGCCAAGAGAGCCACAGGTGTATACCCTGCCCCCATCCAGAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGATTCTATCCATCCGACATCGCCGTGGAGTGGGAGTCCAACGGACAGCCAGAGAACAACTATAAGACCACCCCTCCAATGCTGGACTCCGACGGATCCTTCTTCCTGTATTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGAAACGTGTTCTCTTGTTCCGTGATGCACGAGGCCCTGCACAACCACTATACCCAGAAGAGCCTGTCCCTGTCTCCAGGAAAG6G light chain full antibody nucleotide sequence (SEQ ID NO: 39)GACATCGTGATGACCCAGTCCCCAGACTCCCTGGCCGTGTCCCTGGGCGAGCGCGCCACCATCAACTGCCGCGCCAGCGAATCCGTGGATAACGATCGTATTTCCTTTCTGAACTGGTACCAGCAGAAACCAGGCCAGCCTCCTAAGCTGCTCATTTACGCCGCCACCAAACAGGGTACCGGCGTGCCTGACCGCTTCTCCGGCAGCGGTTCCGGCACCGATTTCACTCTGACCATCTCCTCCCTGCAGGCCGAAGATGTGGCAGTGTATTACTGTCAGCAGTCCAAAGAGTTTCCCTGGTCCTTTGGCGGTGGCACCAAGGTGGAGATCAAACGCACTGTGGCTGCACCATCTGTCTTCATCTTCCCTCCATCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCACGCGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACCCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCTCCAGTCACAAAGAGCTTCAACCGCGGTGAGTGC

1. A method for treating a disease characterized by aberrant depositionof a protein in the brain of a subject, comprising administering to thesubject an effective amount of an antibody comprising threecomplementarity determining regions (CDRs) from a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:1, and three CDRsfrom a light chain variable region comprising the amino acid sequence ofSEQ ID NO:2, wherein the antibody specifically binds to a β-amyloidpeptide, and wherein the disease is cerebral amyloid angiopathy.
 2. Themethod of claim 1, wherein the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:1 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:2.3. The method of claim 2, wherein the antibody comprises a heavy chainpolypeptide comprising the amino acid sequence of SEQ ID NO:11, and alight chain polypeptide comprising the amino acid sequence of SEQ IDNO:12.
 4. The method of claim 1, wherein the antibody comprises a heavychain polypeptide as comprised in vector pDb.9TL.hFc2a deposited as ATCCNo. PTA-6124.
 5. The method of claim 4, wherein the antibody comprises alight chain as comprised in vector pEb.9TL.hK deposited as ATCC No.PTA-6125.
 6. The method of claim 1, wherein the antibody comprises alight chain as comprised in vector pEb.9TL.hK deposited as ATCC No.PTA-6125.
 7. The method of claim 1, wherein the antibody comprises an Fcregion that is not N-glycosylated or has an N-glycosylation pattern thatis altered with respect to a native Fc region.
 8. The method of claim 1,wherein the antibody comprises an Fc region comprising a mutation withina N-glycosylation recognition sequence, whereby the Fc region is notN-glycosylated.
 9. The method of claim 1, wherein the antibody comprisesan Fc region of a human heavy chain IgG2a comprising the amino acidmutation from alanine to serine at position 330 and from proline toserine at position 331, wherein the amino acid position is based onKabat numbering with reference to human wildtype IgG2a sequence.
 10. Themethod of claim 1, wherein the antibody comprises an Fc region havingimpaired effector function.
 11. A method for treating a diseaseassociated with aberrant deposition of β-amyloid in a subject,comprising administering to the subject an effective amount of anantibody that specifically binds to a β-amyloid peptide or an aggregatedform of a β-amyloid peptide, wherein the antibody comprises an Fc regionwith a variation from a naturally occurring Fc region, wherein thevariation results in impaired effector function, and further wherein theantibody comprises a heavy chain variable region comprising: (a) a CDR1comprising the amino acid sequence of SEQ ID NO:3; (b) a CDR2 comprisingthe amino acid sequence of SEQ ID NO:4; and (c) a CDR3 comprising theamino acid sequence of SEQ ID NO:5; and a light chain variable regioncomprising: (d) a CDR1 comprising the amino acid sequence of SEQ IDNO:6; (e) a CDR2 comprising the amino acid sequence of SEQ ID NO:7; and(f) a CDR3 comprising the amino acid sequence of SEQ ID NO:8; whereinthe disease is cerebral amyloid angiopathy.
 12. The method of claim 11,wherein the administration of the antibody with the variation in the Fcregion causes less cerebral microhemorrhage than administration of anantibody without the variation.
 13. The method of claim 11, wherein thesubject is a human.
 14. The method of claim 11, wherein the antibody isa monoclonal antibody.
 15. The method of claim 11, wherein the antibodyis a human antibody.
 16. The method of claim 11, wherein the antibody isa humanized antibody.
 17. The method of claim 11, wherein the Fc regionof the antibody is not N-glycosylated or has an N-glycosylation patternthat is altered with respect to a native Fc region.
 18. The method ofclaim 11, wherein the Fc region of the antibody comprises a mutationwithin the N-glycosylation recognition sequence, whereby the Fc regionis not N-glycosylated.